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Charge-reversal nanocarriers: An emerging paradigm for smart cancer nanomedicine
Journal Pre-proof Charge-reversal nanocarriers: An emerging paradigm for smart cancer nanomedicine
Mingzhen Zhang, Xiaoxiao Chen, Chao Li, Xian Shen PII:
S0168-3659(19)30743-6
DOI:
https://doi.org/10.1016/j.jconrel.2019.12.024
Reference:
COREL 10067
To appear in:
Journal of Controlled Release
Received date:
17 October 2019
Revised date:
13 December 2019
Accepted date:
14 December 2019
Please cite this article as: M. Zhang, X. Chen, C. Li, et al., Charge-reversal nanocarriers: An emerging paradigm for smart cancer nanomedicine, Journal of Controlled Release (2019), https://doi.org/10.1016/j.jconrel.2019.12.024
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© 2019 Published by Elsevier.
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Charge-reversal nanocarriers: an emerging paradigm for smart cancer nanomedicine
Mingzhen Zhanga,c# *, Xiaoxiao Chenb# , Chao Lia, Xian Shena,c*
The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou
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a.
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Medical University, 109 Xueyuan West Road, Wenzhou, Zhejiang 325027, P.R.
The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang
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b.
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China
c.
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325000, P.R. China
WMU-Monash University BDI Alliance in Clinical & Experimental Biomedicine,
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Wenzhou Medical University, Wenzhou, Zhejiang 325027, P.R. China
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*Corresponding authors
Email: [email protected] (M.Z.); [email protected] (X.S.)
# These authors contributed equally to this paper.
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Abstract
The surface charge of nanoparticles (NPs) plays an essential role in determining their biological properties both in vitro and in vivo. In view of the complex features associated with the biological or physiological microenvironment of solid tumors,
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such as electrostatic interactions between NPs and serum components, cellular
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membrane, or intracellular organelles, drug- loaded NPs (or nanocarriers) should
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intelligently accommodate such unique extra- or intracellular microenvironment in
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order to achieve maximum therapeutic and/or diagnostic efficacy. To that end, the
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surface charge of nanocarriers needs to be readily converted at the target site by means of charge reversal, i.e., conversion from anionic to cationic, or vice versa,
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depending on specific microenvironment. In such a manner, the payloads could be
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efficiently released at the desired tumor site. This review discusses 1) the
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physicochemical aspects related to long-circulating nanocarriers for systemic applications; 2) the recent progress in charge-reversal nanocarriers, which are loaded with drugs, nucleic acids, proteins or imaging agents and triggered by various biological signals (i.e., pH, redox, ROS or enzyme) associated with tumor microenvironment, with an emphasis on those induced by acidic tumoral pH; and 3) the perspectives of charge-reversal nanocarriers regarding thorough investigations on how the chemical structure of charge-reversal moiety temporally affects the responsiveness of the resulting nanocarriers toward the rational design of precision cancer nanomedicine. 2
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Keywords : polymeric micelles; drug delivery; gene delivery; nanomedicine;
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charge-conversional polymers; block copolymer micelles
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1. Introduction Over the past three decades or so, numerous drug delivery systems have been developed and are under intensive investigations as cancer nanomedicine, several of which have been approved for clinical applications while many others are still in different stages of clinical trials. Unfortunately, the overall clinical outcomes of
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cancer nanomedicine is not satisfactory in terms of response rates and survival times
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[1, 2], which has triggered reviews regarding nanoparticle (NP)-based cancer therapy
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Such failures could be associated with the significant discrepancy in the amount of
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NPs between at the site of administration and at the site of action [3].
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The advantage of stealthy drug nanocarriers, realized by the incorporation of a poly(ethylene glycol) (PEG) shell on the nanocarrier surface, has been fully validated
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over the past few decades. PEG is neutral, biocompatible and non- immunogenic and
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has been approved by FDA for clinical applications. In particular, PEG has been
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extensively used for constructing cancer nanomedicine [4-6]. Due to the presence of a PEG shell layer on the NP surface, it has been evidenced that PEG was greatly beneficial for prolonged circulation time and enhanced accumulation at tumor site, owing to the EPR effect [6, 7]. However, it has also noticed that the neutral PEG layer was not optimal for cellular uptake given that cell membrane is negatively charged. Therefore, it has been conceived that an ideal nanocarrier should concurrently exhibit the following favorable features: 1) long circulation time and minimal clearance by the reticulo-endothelial system (RES) while in the blood stream; 2) efficient cellular uptake by tumor cells upon arrival at the tumor milieu; and 3) efficient release of 4
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payloads at the targeted intracellular compartment. The emergence of precision medicine for cancer therapy and/or diagnosis can greatly help address the prior challenges encountered for polymeric cancer nanomedicine. With the recent advances in polymer chemistry and by leveraging a wealth of knowledge in tumor biology, it is highly desirable to develop new strategies
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toward intelligent drug nanocarriers (NCs) by which nanocarriers can exhibit
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favorable physicochemical features to accommodate different physiological or
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biological environments, either in the bloodstream, at the tumor milieu, or inside the
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tumor cells, to maximize their biological functions, and eventually therapeutic
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outcomes. The difference in certain biological signals (i.e., pH, redox, ROS and enzyme) existing between the above- mentioned biological microenvironments can be
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utilized as a triggering stimulus toward smart drug nanocarriers, which can respond to
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the stimulus in a site-specific manner and thus exhibit promising propensities toward
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cancer nanomedicine with a potential for clinical applications. Owing to the recent advances in the physicochemical characterization of NPs, it has been demonstrated that the intrinsic features of NPs, such as size, shape and surface charge, play a critical role in determining their biological functions both in vitro and in vivo [8-16]. Furthermore, increasing understanding about tumor biology, in particular, about the tumor microenvironment (TME), could allow for thorough elucidation of the relationship between the physicochemical properties and the biological properties of NPs. In this review, we will focus on how the surface charge of NP (or nanocarrier) interplays with diverse physiological or biological 5
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microenvironment of solid tumors (i.e., intra- or extracellular microenvironment) and how the surface charge of NP plays an essential role in directing the rational design of therapeutic and/or diagnostic agent- loaded NPs toward personalized cancer nanomedicine. First, we provide an overview about the relevant physicochemical foundations of long circulating nanocarriers for systemic delivery of therapeutic
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and/or diagnostic agents, followed by a discussion of different strategies for
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fabricating smart charge-reversal nanocarriers responsive to unique biological signals,
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including pH, redox, ROS and enzyme, in the tumor intra- or extracellular
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microenvironment. The charge-reversal strategy is advantageous for enhanced
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therapeutic and/or diagnostic efficacy and thus may provide a useful solution to
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facilitate the clinical translation of relevant cancer nanomedicine in the future.
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2. Charge-reversal nanocarriers: a powerful solution to address the polycation
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dilemma in the systemic applications of NPs In order to fully understand how the surface charge of NP plays a key role during its journey from the site of administration to the site of action, it is of great importance to have a basic understanding about how the surface charge influences the bio- function of NP in the context of systemic drug delivery, thus providing a rationale for the construction of smart NP by utilizing a novel concept, namely charge reversal. It has been demonstrated that NPs with positive surface charge show significantly higher affinity with negatively charged cell membranes, which is essential for enhanced membrane penetration and cellular uptake [17-20]. Stellacci et al. provided 6
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a valuable summary about how the surface properties (i.e., size, surface charge and surface ligand) of NPs influence the interactions between NPs and cells [17]. By providing insights into NPs of various surface properties, efforts in mechanismic elucidation were actively pursued. It was concluded that surface properties of NPs play a critical role in modulating their interactions with cells. Chen et al. showed that
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more positively charged hydroxyapatite (HAp) NPs could readily penetrate into
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osteoblast cells compared with negatively charged HAp NPs with similar size and
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shape, which was attributed to the attractive or repulsive interaction between cell
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membranes and different charged NPs [18]. Mukherjee et al. studied how gold NPs
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(AuNPs) of various surface charges interacted with plasma membrane of benign or malignant cells [19]. They found that the surface charge of AuNPs plays a
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intracellular events.
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determining role in modulating membrane potential and subsequent downstream
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On the other hand, positively charged NPs can induce significant side effects, in particular in systemic applications, i.e., strong interactions with serum components in the blood and non-specific cytotoxicity against normal cells [19, 21], resulting in rapid clearance, hemolytic side effect and toxicity to normal tissues [22, 23]. In this sense, positively charged nanocarriers are not suitable for in vivo applications. Thus, the development of charge reversal or zwitterionic NPs, which display neutral or negatively charged in the bloodstream but reverse to positively charged upon at the tumor microenvironment, are highly desirable for the systemic delivery of NPs loaded with therapeutic and/or diagnostic agent to cancerous tissues. Due to the site-specific 7
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negative-to-positive charge reversion taking place on the NP surface at the mildly acidic tumor milieu or inside the acidic endo/lysosomal compartments, significantly improved cellular uptake by tumor cells or efficient endosomal escape would be realized while the long circulating, minimal non-specific toxicity features of stealthy NPs are preserved in the negatively charged status while in the bloodstream [24].
Nanocarrier
reversal
composition
Payload
25-28
pH
IC
29-30
pH
IC
35, 53
Drug
pH
IC and TME
51
Drug
pH
TME
52
Drug
pH
36
Pr
Drug
pH
Drug Drug
Enzyme pH
TME TME
62 37
Hybrid micelles
siRNA
pH
IC
80-83
Gold NPs
siRNA
pH
TME
96
Upconversion NPs
PS
pH
TME
106
MSNs
Drug
pH
TME
113
Liposomes
Drug
pH
TME
124, 125
Polymer micelles
Drug Drug
ROS Enzyme
IC IC
70 76
Nanogels
Positive-toNegative
al Drug
IC IC，TME
Polymer-drug conjugates
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Positive
charge reversal
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Negative-to-
Ref.
IC
pDNA Polymer micelles
Site of action for
pH
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Protein
Stimulus
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Type of charge
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Table 1. Representative examples of charge-reversal nanocarriers with different compositions and triggering mechanisms
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IC: Intracellular compartments TME: Extracellular tumor microenvironment PS: Photosensitizer MSNs: Mesoporous silica nanoparticles
3. Charge reversal from anionic to cationic As mentioned previously, the presence of various site-specific biological stimuli 8
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(i.e., pH, redox, ROS, enzyme or a combination of two) could be utilized to trigger charge reversal of NPs. Among these stimuli, a pH gradient between the physiological and the extracellular environment of solid tumors is one of the most frequently used triggering signals. The pH value of the physiological environment is around 7.4 whereas the extracellular environment of solid tumors typically features a pH value
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between 6.4-6.8. Furthermore, endo/lysosomal compartments display a more acidic
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pH value, i.e., 4.5-5.5. The presence of such pH gradient between the extracellular
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and intracellular environments of solid tumors indeed endows a unique opportunity
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for the precision design of smart drug delivery systems targeting the tumor site.
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Therefore, by taking advantage of such site-specific pH values, it is feasible to develop pH-responsive drug delivery systems which can effectively circ umvent biological
barriers
of
the
tumor
extra-
and/or
intra-cellular
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complex
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microenvironment and intelligently release payloads to the targeted locations inside
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the cancer cells to maximize therapeutic outcome. In a pH-induced negative-to-positive charge-reversal strategy, nanocarriers composed of or decorated with a biocompatible polymer possessing pH-dependent charge-reversal moieties are fabricated. These moieties exhibit negatively charged at physiological pH while becoming positively charged at acidic pH, either in the extracellular microenvironment of solid tumors or inside the acidic endo/lysosomal compartments. 3.1.Charge reversal triggered by pH Typically, negative-to-positive charge reversion can be realized through the 9
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pH-dependent transformation of amides into amines occurring on a polymer with charge-reversal moieties. For instance, nanocarriers prepared with a charge-reversal polymer exhibit net negative surface charge in the blood stream but undergo negative-to-positive conversion at the tumor site. In some cases, such nanocarriers further display membrane disruption capability when inside the acidic endo/lysosomal
Charge reversal triggered by acidic endosomal/lysosomal compartments
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3.1.1.
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compartments, owing to a proton sponge effect.
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Kataoka and coworkers reported pioneering investigations in which they used
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different maleic anhydride (MA) derivatives to modify the end group of a protein (i.e.,
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equine heart cytochrome c, CytC) or the side chain of homo- or copolymer of polypeptide, such as polyaspartate (PAsp), PEG-polyaspartate (PEG-PAsp) or
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polylysine to construct pH- labile charge-reversal polymeric micelles for the
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intracellular delivery of proteins [25-28] and pDNA [29, 30]. In order to address the
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challenges encountered in protein delivery, i.e., insufficient charge density of protein to form stable protein/polymer complexes, in one study, they first modified the lysine units of a model protein, CytC, with maleic anhydride (MA), i.e., citraconic anhydride or cis-aconitic anhydride, to respectively produce citraconic amide or cis-aconitic amide and to reversibly increase the anionic charge density of the precursor CytC [26]. It was found that by selecting the number of carboxyl group on the MA, i.e., citraconic anhydride (with 2 COOH) or cis-aconitic anhydride (with 3 COOH), the charge density of the MA- modified CytC was increased in accordance with the number of COOH groups. The charge density of the modified CytC was respectively 10
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increased to -510 Da and -320 Da per charge for citraconic acid and cis-aconitic acid, which is significantly enhanced when compared to the unmodified CytC (i.e., +1391 Da per charge). The resulting anionic CytC was then complexed with a cationic polymer,
namely,
PEG-poly{N-[N’-(2-aminoethyl)-2-aminoethyl]aspartamide}
(PEG-PAsp(DET)) copolymers, to form polyion complex (PIC) micelles. Upon
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internalization into cells and being transported inside the acidic endo/lysosomes,
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maleic amides on the CytC underwent pH-dependent cleavage to expose primary
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amines, thus achieving charge reversal from negative to positive. Thus, charge
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repulsion took place between CytC and the DET-containing polymer, both bearing
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positive charges. Consequently, CytC was expelled from the PIC micelles and released into cytoplasm.
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Moreover, instead of decorating proteins with charge reversal moieties, Kataoka
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also used charge-reversal MA derivatives (i.e., cis-aconitic and citraconic anhydride)
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to modify a polycation, i.e., PAsp(DET), in order to achieve net negative charge. The resulting polymer was used to complex with positively charged lysozyme to form PIC micelles. Likewise, the aconitic amide moiety within the PIC micelles underwent negative-to-positive conversion, which led to the repulsion of lysozyme from the PIC micelles and released to cytoplasm [27].
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Figure 1. Preparation of charge-reversal PIC micelles containing CytC derivatives and PEG–PAsp(DET). a) Citraconic anhydride (or cis-aconitic anhydride/succinic anhydride). Reproduced with permission from Ref. [26]. Copyright 2009, John Wiley
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& Sons.
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In addition to their applications in protein delivery, charge-reversal PEG-based
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copolymers were also widely used to construct non- viral vectors for pDNA delivery
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[29]. It was found that for polycation-based non-viral gene delivery systems, increasing amine density of the polycation or a higher N/P ratio is favorable for membrane destabilization but it induces adverse effect (i.e., cytotoxicity and immune response) due to non-specific interaction with negatively charged serum proteins in the blood. A charge reversal polymer has demonstrated to effectively address the above dilemma. For example, a tertiary system, consisting of pDNA, pAsp(DET) and a charge-reversal polymer pAsp(DET-Aco), was used to delivery pDNA to cells. Poly(aspartic acid) having DET as a flanking group on the side chain (PAsp(DET)) is a cationic polymer and displays endosomal disrupting capability conferred by the 12
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DET moiety. When it was modified with a MA derivative, i.e., aconitic anhydride (Aco), pAsp(DET-Aco) was obtained. As discussed above, aconitic amide undergoes pH-dependent cleavage while at acidic endo/lysosomal compartments and results in unleashing of DET groups, which subsequently exerts endosomal membrane disruption and facilitates PIC micelles to escape from the endosomes. On the other
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hand, due to the negative-to-positive charge conversion taking place for the Aco
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moiety, the cationic PAsp(DET) repels the positively charged pAsp(DET)/pDNA
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polyplex NPs. More importantly, owing to the charge reversal- induced synergic effect,
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efficient endosomal escape was observed for the polyplex NPs, presumably benefited
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from the proton sponge effect conferred by the DET moiety. It is evidenced that as pH value was gradually decreased from neutral to acidic,
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the DET group underwent two-step protonation and exhibited proton sponge effect,
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facilitating NC escaping from the late endosomes or lysosomes. For example, when
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the DET end group of PEG-PAsp(DET) is reacted with citraconic anhydride, it is changed to citraconic amide. Quantification of amine concentration was performed, which shows that the amide group degraded in the acetate buffer (pH 5.5) within 1 h while 60% of the amide groups remained intact at pH 7.4 during a period of 5 h. In contrast, no degradation occurred for the negative control, where a non-pH- labile succinic anhydride was used to react with DET.
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(a)
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(b)
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Figure 2. (a) Charge-reversal ternary polyplexes with endosome-disrupting function; (b) Synthesis of PEG-PAsp(EDA-Cit). Reproduced with permission from Ref. [29] and Ref. [27], respectively. Copyright 2008, John Wiley & Sons; Copyright 2007,
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American Chemical Society.
In the context of intracellular delivery of anti-cancer drugs using NPs, the
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anti-cancer drug, such as camptothecin (CPT), doxorubicin (Dox) and cisplatin, needs
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to interact with DNA located in the cell nuclei to induce cell apoptosis [31-34]. In
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order for the drug to diffuse into the nuclei, it also needs to overcome multiple barriers, which requires that the drug-loaded NPs adapt themselves to the changing biological environments during their journey to the cell nuclei. Shen
and
coworkers
prepared
pH-dependent
polymeric
micelles
and
polymer-drug conjugates for nuclear drug delivery [35, 36] by utilizing a charge-reversal strategy.
In one
study,
polycaprolactone-b-polyethyleneimine
(PCL-b-PEI) copolymer was synthesized, in which part of the primary and secondary amine groups of PEI were masked with amides by using 1,2-cyclohexanedicarboxylic anhydride, i.e., PCL-b-PEI/amide [35]. It was found that majority of these amide 14
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groups promptly hydrolyzed at pH 5-6, while only 50% hydrolyzed at pH 7.4 after 60 h. Such observation was confirmed by zeta potential measurements, in which the resulting PCL-b-PEI/amide micelles maintained a zeta potential of ~ -20 mV at pH 7.4 over a period of over 60 h while the value gradually increased to be slightly positive at pH 6, and further reached +50 mV at pH 5, clearly demonstrating the
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occurrence of a negative-to-positive conversion. As shown in the cellular
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internalization using flow cytometry, the Dox- loaded charge-reversal NPs showed
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faster cellular internalization at pH 6 than at pH 7.4, presumably due to the
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regeneration of positive charges at pH 6 which consequently promoted the cellular
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internalization via electrostatically adsorptive endocytosis. In addition, intracellular trafficking and nuclear localization of Dox- loaded charge reversal NPs was performed
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on SKOV-3 cells using confocal laser scanning microscopy (CLSM), which
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demonstrated that charge reversal improved affinity with the nuclear membrane at 8 h
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post-incubation, followed by NPs entry into the nuclei of cancer cells. Consequently, significant cytotoxicity was observed, as evidenced by dramatically decreased IC 50 . In another study, a polymer-drug conjugate, consisting of poly(L- lysine) (PLL) and camptothecin (CPT), was fabricated for the nuclear delivery to cancer cells [36]. In order to minimize the cytotoxicity induced by the cationic PLL while enhancing cellular uptake efficiency, the following strategy was employed. First, part of the PLL side chain was modified using 1,2-dicarboxylic cyclohexene anhydride (DCA) to form pH-sensitive amides and thus to mask the positive charge. The PLL chain could be regenerated upon at the acidic extracellular microenvironment of tumor. Second, 15
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CPT was conjugated to PLL using a redox-sensitive linker (i.e., disulfide bond), which is responsive to the significantly augmented glutathione concentration in the intracellular environment but remains stable in the extracellular environment. Third, a folic acid (FA) ligand was introduced to PLL to achieve enhanced cellular uptake on FA-overexpressing cancer cell lines. The results show that the negative-to-positive
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charge reversal induced significant enhancement in the cytoxocity of CPT, likely due
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to increased dose of CPT released in the nucleus, as compared to the case in which
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drug resistance was typically the observed for CPT.
Figure 3. The structure of targeted charge-reversal PLL conjugate and tumor acidity-triggered charge reversal (a) and nuclear drug delivery (b). Reproduced with permission from Ref. [36]. Copyright 2009, John Wiley & Sons.
3.1.2.
Charge reversal induced by mildly acidic extracellular microenvironment of
solid tumors
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The vicinity of solid tumors is considered as a first line of barriers for NPs to enter tumor tissues. It has been found that the tumor extracellular microenvironment displays mild acidity, i.e, acidic pHe, compared to the neutral conditions in the bloodstream. Therefore, such pH difference could be utilized as a useful stimulus for charge reversion.
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Wang and coworkers proposed to mask the terminal amines of poly(2-aminoethyl
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methacrylate hydrochloride) (PAMA) with 2,3-dimethylmaleic anhydride (DMA)
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such that amines were converted into amides. The resulting polymer was used to
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fabricate pHe-triggered charge-reversal nanogels [37], of which the amide bond was
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stable at neutral and alkali pH values, but hydrolyzed promptly in response to acidic tumor pHe and recovered the positive-charged amines in the negatively charged
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PAMA-DMA nanogel. Such charge reversion was verified by zeta potential and 1 H
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NMR measurements. As a result of charge reversion at pH 6.8, the zeta potential
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increased significantly, from negative to neutral, within a short period of time (i.e., 35 min) and continued to increase for a prolonged time. In contrast, the zeta potential increased much more slowly at pH 7.4. In the case of succinic anhydride- modified PAMA nanogel, which is a non-charge reversal control, no charge reversion was observed. Furthermore, CLSM and flow cytometry were employed to investigate the cellular internalization of nanogel at pH 7.4 and 6.8, which led to significantly different observations. Remarkable internalization was observed for the nanogel at pH 6.8, as evidence by dramatic fluorescence in the cytoplasm. Howeve r, the nanogel was primarily stick to the cell membrane at pH 7.4. The difference can be explained 17
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by the fact that the negative-to-positive conversion enhances the interactions between the positively charged nanogel and the cell membrane, leading to enhanced cellular internalization. The impact of charge reversal was further corroborated with protein absorption with BSA, in which minimal protein absorption was observed at pH 7.4 while strong absorption occurred at pH 6.8 for the charge-reversal nanogel.
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For the Dox- loaded nanogel, a significantly different drug release trend was
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observed. Charge reversion resulted in accelerated Dox release, dependent on acidity,
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due to decreased interaction between the positively charged Dox and the
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PAMA-DMA nanogel with increasingly protonated COOH groups and the resultant
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positive charges resulting from charge reversal. Overall, Dox- loaded charge-reversal
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nanogels exhibited significantly improved cytotoxicity.
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3.1.3. Charge reversal induced by stepwise response to increasing acidity from
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extracellular tumor microenvironment to endo/lysosomal compartments In addition to biological barriers encountered during the course of intracellular delivery, multidrug resistance (MDR) has also been widely recognized as one of the most challenging and common barriers in the context of cancer chemotherapy due to efflux
of chemotherapeutic
P-glycoproteins (Pgp),
drugs
from cytoplasm by the overexpressed
leading to dramatically reduced therapeutic efficacy [32, 38,
39]. Therefore, bypassing the MDR mechanism would be a critically important for the nuclear delivery of anticancer drugs. One useful strategy to address this challenge is to introduce nuclear localization signal (NLS) to drug delivery systems, such as a TAT 18
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peptide, a cationic peptide derived from human immunodeficiency virus (HIV) [40]. TAT peptide has found extensive applications in transporting various cargos from cytosol to nuclei [41-45], i.e., nucleic acids [41-43], proteins [44-46] and nanoparticulate delivery systems [47-49], in that it can be recognized by nuclear pore complexes (NPC). Therefore, TAT peptide should be helpful in circumventing the
f
Pgp-associated MDR pathway.
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Drug delivery systems, consisting of charge-reversal polymers or based on
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polymer-drug conjugates, have demonstrated superior propensities as intracellular
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drug delivery systems to solid tumors. In general, the negative-to-positive charge
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reversal, realized by masking the positive charges on the drug carrier surface using a maleic anhydride derivative, takes place at the mildly acidic extracellular
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microenvironment of solid tumors. Such strategy has demonstrated to be useful in
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addressing the dilemma associated with long circulation time and minimal
carboxylic
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non-specific interactions between drug carriers and serum component. When the group
underwent pH-dependent
hydrolysis
in
the extracellular
microenvironment, the positive charges were readily regenerated, which resulted in remarkably enhanced cellular uptake and cytotoxic ity on cancer cells. It was also recognized that upon internalization via endocytosis, these single pH-responsive drug delivery systems need to overcome additional pH-associated biological barrier located inside organelles, namely, endo/lysosomes, in order to readily release drug payload. As mentioned above, the pH value inside the endo/lysosome compartments is 4.5-5.5, which is significantly lower than that in the extracellular microenvironment and thus 19
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requires a second mechanism by which the linker between the drug and the polymer undergoes prompt cleavage at endo/lysosomal pH to facilitate drug release. To concurrently achieve the above goals, Wang and coworkers further prepared a dual pH-responsive polymer-drug conjugate, consisting of polyphosphoester (PPC) and Dox [50]. The polymer was first conjugated with Dox through a hydrazone (Hyd)
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linker and was then end-modified with a maleic anhydride derivative (i.e., DMA) to
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produce amide bonds. The resulting hydrazone and maleic amide linkages are acid
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labile and can respond to mildly acidic and endo/lysosomal pH value, respectively.
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The pH-dependent cleavage of maleic acid amide and hydrazone linkage was
Cellular
internalization
and
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confirmed by 1 H NMR and the release of Dox at desired pH range, respectively. intracellular
trafficking
were
performed
on
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MDA-MB-231 cells using flow cytometry and CLSM, respectively, which showed
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enhanced uptake of PPC-Hyd-Dox-DA NPs and efficient release of Dox from the NP
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release, and improved presence in the cell nuclei, as compared to controls. These results are attributed to the dual responsiveness toward a pH gradient, i.e., pHe 6.5 for the extracellular tumor microenvironment and pH 4.5-5.5 for acidic endo-/lysosomes. Moreover, the dual pH-responsive propensity exhibited great potential in inhibiting cancer stem cells, clearly demonstrating the advantage of a dual pH-responsive drug NC.
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Journal Pre-proof Figure 4. Chemical structure of PPC-DA. Reproduced with permission from Ref. [50]. Copyright 2011, American Chemical Society.
As mentioned previously, bypassing the Pgp-associated multidrug resistance (MDR) is critical to achieve enhanced therapeutic efficacy in cancer chemotherapy using a drug delivery system. Given that the cationic TAT peptide, a type of cell
f
penetrating peptides (CPP), has shown to be helpful in circumventing the
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Pgp-associated MDR mechanism, the introduction of TAT peptide to a charge-reversal
a
polypeptide,
namely
Poly(L- lysine)-block–poly(L- leucine)
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synthesized
pr
drug delivery system could be advantageous. To this end, Zhang and coworkers
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(PLL-b-PLLeu) [51]. The lysine residue was amidized with dimethylmaleic anhydride (DMA) derivative to construct negatively charged micelles, possessing nuclear
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localization and charge reversal properties. Moreover, the TAT peptide was amidized
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with succinyl chloride (SA), an acid-degradable moiety, to mask its positive charge
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and cell-penetrating features and was then conjugated with the polypeptide. The resulting polypeptide, modified with two acid- labile moieties responsive to two different pHs, was used to fabricate Dox-loaded PLLeu-PLL(DMA)-TAT(SA) micelles. In view of the presence of two pH- labile moieties within the same micelles, the micelles underwent sequential charge reversal, first for the DMA moiety at mildly acidic microenvironment and then for the SA moiety in the endo/lysosomal compartments. Consequently, in addition to TAT being regenerated to confer cell-penetrating capability, micelles also became positively charged, which led to significantly increased delivery of Dox to the cell nuclei, as evidenced from the flow 21
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cytometry measurement and CLSM observation. Thus, the notion of dual pH-responsive polymers or polymer-drug conjugates has clearly demonstrated the advantages of realizing dual negative-to-positive charge reversals at two pH values within the same drug carrier, first in the tumor microenvironment (pH 6.8) and then in the endo/lysosomal compartments (pH
f
4.5-5.5). Such strategy not only extends circulation time and minimizes non-specific
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interaction with serum components, it also facilitates the nuclear delivery of
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anticancer drug, as demonstrated by significantly increased cytotoxicity to cancer
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al
Pr
e-
cells.
Figure 5. Schematic illustration of (A) the self-assembled polypeptide micelles stepwise responding to mildly acidic tumor tissues through a charge reversal process and to more acidic endosomes to reactivate the nuclear location function of TAT peptide; (B) cellular uptake of nanoparticles and nuclear delivery of drugs. Reproduced with permission from Ref. [51]. Copyright 2015, John Wiley & Sons.
3.1.4
Charge reversal as a means to achieve size-shrinkable nanoparticles 22
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It has been revealed that size of NPs plays a critical role in the biological property in the context of polymeric cancer nanomedicine. Kataoka et al. demonstrated that the size of polymeric micelles strongly affects the biological properties, such as biodistribution and tumor penetration in xenograft pancreatic tumor model [8]. The results showed that although NPs having a size in the sub-100 nm range is optimal to
f
achieve long blood circulation and enhanced tumor retention via the EPR effect,
oo
smaller size is critical for deep tumor penetration, thus enhanced antitumor efficacy.
pr
In order for chemotherapeutic agents (such as Dox, CPT and cisplatin) to exert
e-
biological functions (i.e., inducing apoptosis), such agents must enter into cell nucleus
Pr
and interact with DNA molecules located there. In addition to the aid of NLS, NPs of small size become an essential factor in determining whether they could efficiently
al
enter the nucleus. In other words, small-sized NPs are required. In order to
rn
concurrently address the conflicting size requirements required for the EPR effect (i.e.,
Jo u
sub-100 nm) and nuclear entry (i.e., sub-50 nm), an ideal strategy would be to design a nanocarrier which exhibits sequential size shrinking, depending on the stage of particle transportation or on the particle location, i.e., from blood circulation to location in the cytoplasm or inside the endo/lysosomes. In accordance with such a notion, a NC system should contain two different types of functional gro ups in order to achieve two-stage size shrinking, taking place respectively in the extracellular and intracellular environment. Shen
and
coworkers
used
a
maleic
anhydride
derivative
(i.e.,
1,2-dicarboxylic-cyclohexene anhydride, DCA) to mask majority of the positively 23
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charged amino termini on PAMAM-G5 to confer negative charges, which is beneficial for prolonged circulation time and minimal non-specific interaction with serum component in the blood [52]. Upon at the acidic tumor microenvironment (TME), hydrolysis of amides led to regeneration of NH2 termini as well as positive charges, thus facilitating cellular uptake by cancer cells. Moreover, the remainder of the
f
terminal NH2 groups was modified with folic acid ligand and thiolated CPT,
oo
respectively. The advantage of thiolated CPT is that when the NC is internalized into
pr
cells, the disulfide linker breaks up and CPT can be released from the NC due to
e-
significantly elevated glutathione (GSH) concentration inside the cells. As a
Pr
consequence of such stepwise cleavage of amide and disulfide linkages occurring respectively at extracellular and intracellular environment, dramatic decrease in
al
particle size was achieved, which resulted in enhanced drug delivery to the nucleus,
rn
clearly demonstrative of the merits of a charge reversal strategy.
Jo u
In another study, Huang et al. prepared N-(2-hydroxypropyl) methacrylamide (HPMA)-based nanovehicles constructed via electrostatic interaction between oppositely charged polymers [53]. The anionic component was obtained by modifying the HPMA side chain with charge reversal moieties, i.e., DMA, while the cationic component contained intracellular detachable subgroup (IDS), consisting of Dox and a cell penetrating peptide (CPP), i.e., octaarginines peptide and an NLS (R8NLS). Under physiological conditions, the pristine nanovehicles exhibited neutral surface charge and suitable particle size, thus possessing propensities for prolonged blood circulation and enhanced tumor accumulation through the EPR effect. Upon at the 24
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acidic TME, or first-stage tumor acidity, charge reversal of the anionic component took place. Consequently, the nanovehicles dissociated and transformed into much smaller conjugates, leading to the exposure of the R8NLS species to facilitate cellular uptake. Next, upon localization in the more acidic endo/lysosomal compartment, the nanospecies underwent a second-stage size reduction wherein the IDS detached from
f
the copolymer and further shrank its size to enhance NLS-mediated nuclear pore
oo
complex transport. By means of FRET and CLSM, two-stage size shrinking led to
pr
remarkably enhancement in cellular uptake and nuclear entry of the Dox-containing
Jo u
rn
al
Pr
e-
species, in consistent with the improved anticancer efficacy in vivo.
Figure 6. Schematic illustration of a multi-stage size reduction strategy using HPMA-based polymeric nanovehicle (PNV). i) Temporary shielding of cationic P-DoxR8NLS using charge-reversal P-DMA, which is anionic at neutral pH to prolong circulation. ii) Self-assembly into PNV through electrostatic attraction, leading to size increase and enhanced EPR effect. iii) Acidic tumor extracellular microenvironment results in charge reversal in P-DMA and collapse of PNV into smaller copolymer chains. iv) Exposure of R8NLS enhances internalization into tumor cells. v) Intracellular release of IDS due to acidic hydrolysis of the hydrazone linkage in the acidic endo/lysosomal compartments, leading to a second-stage size reduction. vi) Endo/lysosomal escape and NLS-assisted nuclear targeting. Reproduced 25
Journal Pre-proof with permission from Ref. [53]. Copyright 2015, John Wiley & Sons.
3.2. Enzyme-triggered charge reversal In addition that positively charged nanocarriers exhibit dramatically enhanced cellular uptake and are beneficial to overcome biological barriers toward improved therapeutic efficacy, it has been recognized that the ATP-dependent transcytosis,
oo
f
which transports cargos across the endothelium, plays a vita l role in enhancing the transport of nanocarriers across the capillary wall into tumor tissues. Bae et al. have
pr
demonstrated that cationic nanocarriers can effectively promote adsorption- mediated
e-
transcytosis (AMT) and facilitates nanocarriers to penetrate across multiple cell layers
Pr
[54]. As mentioned above, cationic nanocarriers are not suitable for in vivo
al
applications since they compromise the long circulation property and induce
rn
significant non-specific toxicity. A charge-reversal strategy could be an effective solution to this challenge, in which NPs display different surface charges during blood
Jo u
circulation or in the vicinity of tumor tissues. As extensively discussed above, a pH gradient has been widely utilized to trigger negative-to-positive charge reversal and thus is able to fulfill that goal when nanocarriers are navigated from the neutral physiological environment to the mildly acidic tumor microenvironment or to the acidic endo/lysosomal compartments. However, in the context of luminal and perivascular compartments, the application of a pH gradient is limited not only by the presence of the local neutral environment [55], but also by the fact that mildly acidic microenvironment is so far away from the tumor blood vessel networks (i.e., 100-200 m) to exert an effective pH gradient [56, 57]. 26
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In order to overcome the challenges associated with the pH-triggered charge reversal taking place in the luminal and perivascular regions, a novel strategy was necessary. It has been found that cell surface enzyme, i.e., γ-glutamyl transpeptidase (GGT), can be used to cleave γ-glutamyl bonds, i.e., -glutamylamides. Considering that GGT is overexpressed on the surface of endothelial cells in various human
f
tumors (i.e., liver, cervical and ovarian cancers) [58, 59], GGT could be utilized as a
oo
potential target in the design strategy of cancer therapy and cancer imaging probes [60,
pr
61]. By means of selective cleavage of γ-glutamyl bonds, i.e., -glutamylamides of
e-
glutathione (GSH), GGT can be used to fulfill certain biological functions associated
Pr
with cancer cells.
Based upon these findings, Shen et al. recently proposed to synthesize a polymer-drug
conjugate,
al
GGT-responsive
acrylamide)
of
(PBEAGA)
and
rn
poly(2-(l-γ-glutamyl- l-α-aminobutyrylamino)ethyl
consisting
Jo u
camptothecin (CPT) (PBEAGA-CPT). The conjugate remained negatively charged in the bloodstream to exert prolonged circulation and stealthy properties. Upon contact with the tumor endothelial cells, which features elevated GGT expression level, the conjugate underwent GGT-mediated cationization, namely GGT-catalyzed hydrolysis of -glutamylamide to produce an amino derivative, resulting in negative-to-positive charge reversal to the conjugate [62]. The resulting cationic conjugate was revealed for
efficient
cellular
internalization
and
transcytosis,
allowing
enhanced
transendothelial and transcellular transport. Consequently, improved therapeutic efficacy was expected. As evidenced from the zeta potential measurements, the zeta 27
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potential of the PBEAGA-CPT conjugate changed from negative to positive within 15 h in the presence of 10 U/mL GGT, which was not observed for the control not containing a GGT-sensitive bond. In vitro experiments showed that compared to a control, i.e., GGT-negative fibroblast cells, only the GGT-positive cancer cells displayed significantly increased cytotoxicity. These results were substantiated with
f
cellular internalization pathway and intracellular trafficking studies using 2D cell
oo
culture, as well as with cellular penetration studies using multi-cellular spheroids to
pr
mimic tumor tissues, which demonstrated that the penetration of the GGT-sensitive
e-
PBEAGA-CPT conjugate was through a transcytosis mechanism dependent on the
Pr
caveolae- mediated endocytosis and exocytosis. In vivo experiments revealed the preferential accumulation of PBEAGA-CPT conjugate in the GGT-overexpressed
al
liver tumor site (i.e., HepG2) and dramatically enhanced antitumor efficacy, as
rn
evidenced from the eradicating of large tumors (i.e., subcutaneous HepG2) and extend
Jo u
survival rate of mice bearing orthotopic pancreatic tumors.
28
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Figure 7. Schematic representation and zeta potential characterization of the enzyme-activatable polymer–drug conjugate. Reproduced with permission from Ref. [62]. Copyright 2019, Springer Nature.
4. Charge reversal from cationic to anionic The strategy of stimuli-triggered charge reversion, typically reversion from
oo
f
anionic to cationic, has been widely employed in the rational design of NC as cancer
pr
nanomedicine, and has contributed to significant progress in the development of cancer nanomedicine toward clinical applications. Recently, the concept of
e-
cationic-to-anionic charge reversal has received a great deal of interest in nanocarrier
Pr
design.
al
In the field of non- viral gene vectors, cationic polymer or liposomes have been
rn
extensively used to construct nano-sized polyplexes or lipoplexes to deliver nucleic
Jo u
acid (pDNA or siRNA) payload to cells, where the nucleic acid exerts its biological functions. However, one complication associated with cationic vector is that the positive charge, presented on the dissociated cationic debris, was found to interact and interfere with the intracellular DNA transcription machine, thus leading to non-specific toxicity [63, 64]. To address such a drawback, a new paradigm of non-viral gene vector, which does not rely on the conventional electrostatic attraction between a cationic vector and an anionic nucleic acid to form polyplexes, should be useful since the dissociation of polyplexes between the cationic vector and the anionic nucleic acid generates a large
29
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amount of short cationic debris harmful to DNA transcription machine. Instead, a new class of gene vector would depend on the electrostatic repulsion between two anionic species, i.e., nucleic acid and a charge reversal polymer, to deliver payloads. Such strategy should be a promising alternative in that it can eliminate the production of
f
cytotoxic cationic debris, as is the case for the conventional gene vectors.
oo
4.1.ROS-mediated charge reversal from cationic to anionic
pr
It has been demonstrated that, owing to oncogenic transformation, cancer cells
e-
generate higher levels of intracellular reactive oxygen species (ROS), including H2 O 2 ,
Pr
hydroxyl radical, and superoxide, than normal cells [65, 66]. Thus, such feature of cancer cells could be utilized as a stimulus to mediate efficient intracellular delivery
one
study,
Shen
Jo u
In
rn
have been reported [67-72].
al
of payloads using smart nanocarriers. Several ROS-responsive polymeric nanocarriers
poly[(2-acryloyl)ethyl(p-boronic
et
al. acid
synthesized
a
cationic
benzyl)diethylammonium
polymer, bromide]
(B-PDEAEA), which was modified with a reactive oxygen species (ROS)-sensitive charge-reversal moiety capable of positive-to-negative conversion [70]. Upon at elevated ROS (i.e., H2 O2 ) level inside tumor cells, it could oxidize boronic acid groups by ROS, leading to release of p- hydroxylmethylenephenol (HMP) from the quaternary ammonium [73] and resulting in the formation of tertiary amines. Consequently, it catalyzed the conversion from polyacrylate to anionic poly(acrylic acid) [74]. As a result of hydrolysis of ester group to produce carboxylic acid, i.e., 30
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poly(acrylic acid), the zeta potential underwent positive-to-negative conversion in a certain timeframe, depending on pH value. For instance, it accelerated with decreasing pH value. Due to the ROS-induced charge reversal, anionic nucleic acid was readily released from the polyplexes without interfering with the DNA transcription machine, thus dramatically minimizing the non-specific toxicity. As
f
shown in the gene transfection experiments using a reporting gene, the gene vector
oo
prepared with a charge-reversal polymer (i.e., B-PDEAEA) exhibited significantly
pr
enhanced gene transfection efficiency in cancer cells, but not in normal cells. It was
e-
also substantiated with in vivo experiments, in which a therapeutic DNA (i.e.,
Jo u
rn
al
Pr
pTRAIL) selectively induced apoptosis in subcutaneous A549 tumor-bearing mice.
Figure 8. Reaction oxygen species (ROS)-responsive charge-reversal polymer as non-viral gene delivery system. (a) Structure of B-PDEAEA and its ROS-induced charge reversal. (b) Formation of B-PDEAEA/DNA polyplexes and its ROS-triggered charge reversal to anionic polyacrylic acid. Reproduced with permission from Ref. [70]. Copyright 2016, John Wiley & Sons. 31
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4.2. Enzyme-triggered charge reversion from cationic to anionic In addition to killing cancer cells, chemotherapy was also found to induce adverse effect to fibroblasts by triggering a WNT16B pathway, which is accountable for tumor relapse and poor prognosis of chemotherapy [75]. Thus, it is desirable to design a drug delivery system which selectively exerts damaging effect to cancer cells, but not
oo
f
to fibroblasts. To this end, an esterase-responsive polymer (ERP), based on quaternary amines and possessing ester substitutes, was prepared as non-viral gene carriers [76].
pr
By taking advantage of the dramatic difference in esterase activity between cancer
e-
cells (i.e., HeLa) and fibroblasts (i.e., NIH3T3), the resulting ERP/pDNA polyplexes
Pr
promptly dissociated and released pDNA inside HeLa cells, owing to the
al
positive-to-negative charge reversal induced by the elevated esterase activity, but
rn
barely inside the fibroblasts. Consequently, the released pDNA encoding a cancer suicide gene (i.e., pTRAIL) was able to selectively kill cancer cells while induced
Jo u
minimal damaging effect to the fibroblasts, thus circumventing the WNT16B activation mechanism. The results show that such esterase-responsive gene vector was remarkably effective in inhibiting cancer cells in vitro and in vivo.
5.
Application of charge-reversal polyme rs (CRP) or components in the
fabrication of hybrid or liposomal nanocarriers for cancer therapy or diagnosis 5.1. Inorganic-organic hybrid micelles for siRNA delivery Besides its applications in fabricating polymeric micelles or polymer-drug conjugates, polymers possessing charge-reversal feature, or charge-reversal polymers 32
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(CRPs) are also useful in constructing organic- inorganic hybrid micelles. Given that calcium phosphate (CaP) has been extensively used in biomedical materials, i.e., drug/gene delivery systems, due to its advantages such as excelle nt biocompability, biodegradability and easy preparation [77-79], integration of biocompatible PEG-polypeptide and CaP into one hybrid delivery system, or one hybrid
f
naomedicine, should be highly beneficial for biomedical applications. Kataoka and
oo
coworkers took advantage of the anionic PEG-based CRP and siRNA to fabricate
pr
CRP/CaP/siRNA hybrid micelles [80-83], based on a notion that anionic species could
e-
inhibit the crystal growth of CaP NPs, resulting in the formation of hybrid
Pr
siRNA- loaded nanoparticles. First, together with anionic siRNA, a PEG-based block copolymer, with flanking amine groups on the side chain modified with aconitic
al
anhydride to confer negative charge, was used to complex with CaP to form
rn
CRP/siRNA/CaP hybrid nanocarriers. Such nanocarriers were characteristic of
Jo u
prolonged circulation time and excellent biocompatibility. Upon internalization into pancreatic cancer cell lines, i.e., PanC-1 and BxPC-3 cell lines, via endocytosis, the CRP within the hybrid nanocarriers underwent pH-dependent cleavage of amides and thus recovered the cationic amine moieties with endosomal membrane disrupting capability. Consequently, siRNA nanocarriers could escape from the acidic endo/lysosomal compartments and release siRNA into cytoplasm. Significant gene knockdown against endogenous VEGF was observed on pancreatic cancer cells, demonstrating great potential for siRNA delivery [80, 81]. In addition, the hybrid nanocarriers were used for the systemic siRNA delivery to spontaneous pancreatic 33
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tumors in EL1- Luc/TAg transgenic mice, in which increased accumulation of siRNA- loaded hybrid nanocarriers as well as remarked gene silencing again luciferase was observed.
These studies clearly demonstrated
the benefits of using
al
Pr
e-
pr
oo
f
charge-reversal siRNA/CaP hybrid nanocarriers in RNAi-based therapy [83].
rn
Figure 9. (A) Schematic illustration of hybrid micelles composed of PEG-CCP,
Jo u
siRNA, and CaP. (B) Chemical structure of PEG-PAsp(DET-Aco) (PEG-CCP). (C) Schematic illustration of the cellular delivery of siRNA by PEG-CCP/CaP hybrid micelles. Reproduced with permission from Ref. [81]. Copyright 2012, Elsevier.
5.2. Gold NPs coated with CRP for siRNA delivery Gold nanoparticles (AuNPs) have emerged as a promising delivery system for various payloads into cells, such as small drug molecules [84-88] or nucleic acids (i.e., pDNA and siRNA) [89-96]. Typically, nucleic acids were incorporated with AuNPs through a thiol linkage or electrostatic interaction with the cationic AuNPs. For example, a polycation, such as polyethyleneimine (PEI), was employed to coat onto 34
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the AuNP surface and subsequently bind with the anionic siRNA. Such siRNA- loaded AuNP exhibited strong endosomal escape capacity due to the proton sponge effect conferred by PEI. Unfortunately, low extent of siRNA was released into cytoplasm as a result of the high binding affinity between the AuNP and siRNA. Thus, increasing amount of siRNA is needed in order to achieve significant gene knockdown efficacy,
f
which is optimal for formulation.
oo
To address such a challenge, Liang et al. designed a type of ternary complexes
pr
composed of siRNA, a polycation, and a charge-reversal polymer [96]. Specifically, a
e-
charge-reversal siRNA nanocarrier was prepared using cationic PEI, charge-reversal
Pr
citraconic anhydride- modified poly(allylamine hydrochloride) (PAH-Cit), and mercaptoundecanoic acid-modified AuNPs (MUA-AuNPs) through a layer-by-layer technique.
Consequently,
PEI/PAH-Cit/PEI/AuNPs
were
obtained.
al
assembly
rn
Polyacrylamide gel electrophoresis (PAGE) was performed to determine the amount
Jo u
of siRNA released from the ternary AuNPs. The results showed that increasing amount of siRNA was released with increasing incubation time at pH 5.0 as a result of charge reversion of the Cit moiety in the siRNA/PEI/PAH-Cit/PEI/MUA-AuNP system, which is in contrast to control non-charge-reversal nanoparticles under identical conditions, i.e., siRNA/PEI/PSS/PEI/MUA-AuNPs. Furthermore, in vitro experiments, including gene transfection and siRNA knockdown studies, showed enhanced payload release and gene silencing efficacy due to the charge reversion of PAH-Cit. The advantage of such charge-reversal AuNPs as a siRNAtransfection agent was demonstrated by the observation that the charge-reversal AuNPs display higher 35
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siRNA transfection efficiency than the commercial transfection agents, such as Lipofectamine and PEI.
5.3. Upconversion NPs coated with CRP for diagnosis Photodynamic therapy (PDT) has emerged as a powerful strategy for cancer
f
treatment [97-99]. During the PDT treatment, ROS and singlet oxygen are generated
oo
upon light irradiation of a photosensitizer (PS) molecule at a selective wavelength,
pr
spanning from visible to NIR light. PDT is advantageous in that it can selectively kill
e-
cancer cells around the tumor lesion while minimizing damage to normal tissues.
Pr
More importantly, with the advances in PS synthesis, PDT can now be realized using an NIR light, in comparison to a conventional PDT treatment using visible or even
al
UV light. Thus, PDT using NIR-activation allows for much deeper tissue penetration
rn
and enables treatment of internal or large tumors [15, 72, 100].
Jo u
Upconversion nanoparticles (UCNPs) have demonstrated as promising materials for biomedical applications and in particular, have received tremendous attention as cancer nanomedicine [101-108]. In an upconversional system, absorption of two or more NIR photons leads to emission of a single photon with higher energy or a shorter wavelength. Such unique features can significantly address such issues as auto-fluorescence background, tissue penetration depth, and phototoxicity, which are commonly encountered in the traditional PDT treatment utilizing visible or UV light for excitation. Taking advantage of the merits of NIR-PDT and UCNPs, UCNPs have found tremendous potential in NIR-based PDT treatment of solid tumors [106, 109]. 36
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More recently, the development of smart dual- or multi- modal cancer nanomedicines, has opened up dramatic opportunities for UCNPs-based PDT. These new nanomedicines are advantageous over the conventional single- modal ones and should resolve the common issues of extended circulation and enhanced retentio n. Being able to
respond
to tumor-specific extracellular and/or
intracellular
f
microenvironment, smart UCNPs have shown significant enhancement in treatment
oo
efficacy for the in vivo PDT treatment of cancer in mice models.
pr
Liu and coworkers synthesized Mn2+-doped UCNPs emitting red light at ca. 660
e-
nm with an NIR excitation at 980 nm. The resulting UCNPs were subsequently used
Pr
to activate a photosensitizer, i.e., Chlorin e6 (Ce6), conjugated to cationic poly(allylamine hydrochloride) (PAH) to generate singlet oxygen for killing cancer
al
cells [106]. Ce6 molecules were loaded into the UCNPs through a layer-by-layer
rn
(LbL) approach, in which Ce6-PAH electrostatically complexed with anionic
Jo u
poly(acrylic acid) (PAA) decorated UCNPs. In order to confer negatively charged surface, DMMA modified PAH was used as a terminal coating for the NPs via LbL. The amide bonds underwent pH-dependent hydrolysis upon at the acid TME, which led to positively charged surface and dramatically enhanced cellular uptake of NPs, thus enhancing NIR- induced PDT efficacy in vitro. Due to Mn2+ doping, the UCNPs displayed optical and paramagnetic properties for in vivo dual- modal imaging after intratumoral injection. Overall, the Mn2+-doped UCNPs exhibited significantly enhanced PDT therapeutic efficacy in vivo, owing to the utilization of charge reversion polymer which conferred critical pH-dependent transformation for the 37
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UCNPs while at the tumor microenvironment.
5.4. Mesoporous silica nanoparticles (MSNs) coated with charge-reversal components to deliver anticancer agent Mesoporous silica nanoparticles (MSNs) have emerged as a promising candidate
f
for biomedical applications, in view of their excellent biocompatibility, high internal
oo
pore volume for drug loading, ease of functionalization, among others [110-112].
pr
Several surface modification strategies have been proposed to overcome challenges in
e-
the aspects of prolonged circulation, targetability, minimal non-specific adsorption of
Pr
proteins to the surface, among others. Although modification of MSN surface with zwitterionic species has demonstrated to be a useful approach toward stealth property,
al
such surface decoration may adversely affect the cellular internalization of the
rn
resulting MSNs. Therefore, a new strategy, which could address the above dilemma,
Jo u
is desirable. Chen et al. proposed to synthesize fluorescent MSNs which possessed switchable zwitterionic surface and were capped with polycaprolactone (PCL), which degrades at increased esterase activity inside cancer cells [113]. The resulting MSNs were loaded with an anticancer drug, Dox. Under physiological conditions, the MSNs exhibited excellent anti-biofouling property toward model plasma protein (i.e., bovine serum albumin) and esterase could not reach the PCL gate to trigger cargo release. While at acidic tumor milieu (pH<6.8), charge reversion took place, which diminished the anionic part of the zwitterionic surface and led to positively charged surface to facilitate cellular uptake by cancer cells. At the same time, due to enzymatic 38
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degradation of the PCL cap at the increasing presence of esterase in the MSN pore, intracellular release of Dox was triggered, as evidenced from the presence of free Dox in the nucleus and significantly enhanced cytotoxicity to cancer cells. The results clearly demonstrated that the synergetic effect induced by tumor acidity- induced charge reversal on the MSN surface and enzymatic degradation of PCL at pore gate
f
could effectively overcome the challenge of prolonged circulation time in the
oo
bloodstream and efficient cellular uptake and intracellular release of anticancer drug
pr
in tumor tissue, which is a common challenge in the application of MSNs as drug
Jo u
rn
al
Pr
e-
delivery systems.
Figure 10. Schematic representation of PCL/Dox- loaded fluorescent mesoporous silica nanocarriers with switchable zwitterionic surface and targeted drug delivery to cancer cells. Reproduced with permission from Ref. [113]. Copyright 2016, John Wiley & Sons.
5.5. Charge-reversal liposomes to deliver anticancer agent Liposomes, typically composed of phospholipids, are spherical vesicles of lipid bilayers and an aqueous core within the bilayers, which have demonstrated to be a 39
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safe and effective carrier system to incorporate hydrophobic and hydrophilic therapeutics [114-117]. In particular, liposomes have been widely used to deliver anticancer agents, i.e., small molecule drugs, nucleic acids and proteins, for cancer treatment. In order for better applicability in the systemic delivery of anticancer therapeutics, surface modification of liposomes with biocompatible PEG, or
f
PEGylation of liposomes, can lead to stealth liposomes, which display significantly
oo
improved blood circulation time and enhanced accumulation of the encapsulated
pr
anticancer agents into solid tumors through an enhanced permeability and retention
e-
(EPR) effect [118, 119]. Furthermore, liposomes can be modified with various ligands
Pr
to achieve specific targeting [120-122].
In view of the presence of biological challenges (i.e., improved blood circulation
al
and endosomal escape) during the course of systemic delivery, which could hinder the
liposomes
are
necessary.
To
that
end,
Mo
et
al.
prepared
a
Jo u
smart
rn
effective delivery of payload encapsulated within the liposomes, strategies toward
mitochondrial- targeted liposomal delivery system composed of a zwitterionic oligopeptide (HHG2C 18 -L), which exhibits multi-stage pH response to successively surpass a series of biological barriers in the systemic delivery of an anticancer drug, namely
temsirolimus
phosphatidylcholine,
[123].
The
cholesterol,
resulting and
liposomes a
synthetic
consist lipid
of
soy (i.e.,
1,5-dioctadecyl-L- glutamyl 2- histidyl- hexahydrobenzoic acid, HHG2C 18 ). The lipid carries a pH-cleavable group (i.e., hexahydrobenzoic amide) as a hydrophilic block, two stearyl alkane chains as a hydrophobic block, and two amino acids (i.e., glutamic 40
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acid and histidine) to resemble the structure of natural phospholipids, which confers the resulting HHG2C18-L a multi- stage pH-responsive property, i.e., first to the mildly acidic extracellular tumor microenvironment and then to the acidic intracellular endo/lysosomal compartments. HHG2C18 -L displays negatively charged during blood circulation whereas it exhibits positively charged upon arrival at the mildly acidic
f
tumor microenvironment (i.e., around pH 6.5) due to the presence of zwitterionic
oo
oligopeptide. As a result of charge reversion or first-stage pH response, enhanced
pr
endocytosis took place. While at the acidic endo/lysosomal compartment, the histidine
e-
groups on the positively charged liposomes achieved proton sponge effect to facilitate
Pr
endosomal escape (i.e., second-stage pH response). Note that the pH-dependent hydrolysis, enabled by the pH-cleavable linker (i.e., hexahydrobenzoic amide) and
al
occurring in cytoplasm (i.e., third-stage pH response), was useful in maintaining the
rn
positive charge of HHG2C18 -L after its endo/lysosomal escape. It has been
Jo u
demonstrated that positive charge is critical to facilitate interactions with mitochondria to achieve mitochondria targeting. The results showed that, compared to non-pH-responsive controls, the introduction of charge reversion as well as multi-stage pH-response properties to HHG2C18-L significantly improved the therapeutic efficacy, as evidenced from the in vitro and in vivo experiments. In another study performed by Mo and coworkers [124], they demonstrated that the PEGylated HHG2C18-L also exhibited charge reversal and multi-stage pH responses to the mild acidic TME and the acidic endo/lysosomal compartments, successively. When encapsulated with anticancer agent (i.e., temsirolimus), the resulting 41
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drug- loaded liposomes displayed significantly enhanced tumor inhibition against xenograft renal cancer tumor models, thus validating the effectiveness of applying a
al
Pr
e-
pr
oo
f
charge-reversal strategy to liposomal delivery system.
Figure 11. Schematic representation of smart liposomes with multi-stage pH response
rn
to extracellular tumor microenvironment and intracellular compartments for drug
Jo u
delivery. Reproduced with permission from Ref. [123]. Copyright 2012, John Wiley & Sons.
6. Biosafety issues associated with charge-reversal nanocarriers Although the promising propensity realized by charge-reversal nanocarriers has triggered intense interest among the research community of cancer nanomedicine, the biosafety issues associated with charge reversal at biological or physiological environment are not fully understood yet and thus should deserve careful and systematic investigations. It is known that nanocarriers with positive surface charge are conducive to enhanced cellular uptake but they also induce undesired interaction 42
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with anionic serum components, leading to reticulo-endothelial system (RES) uptake in the liver and spleen (i.e., loss of injected dose), reduced circulation time and significant immune response. Such dilemma can be addressed by employing a charge-reversal strategy. Owing to the introduction of a charge-reversal moiety (i.e., negative-to-positive conversion) to the nanocarrier, the positive surface charge of
f
nanocarrier can be temporarily masked with a charge-reversal component to impart
oo
anionic or neutral feature during the blood circulation, which results in extended
pr
blood circulation time and minimal immune response. Up to date, most studies have
e-
only investigated the systemic toxicity or immune response after the systemic
Pr
administration of charge-reversal nanocarriers into mice. Given the fact that prior to negative-to-positive conversion, smart nanocarriers are neutral or anionic, which
al
usually exhibit excellent biocompatibility, owing to rational design of biocompatible
rn
nanocarriers. It, however, should be noted that upon the negative-to-positive
Jo u
conversion occurring at the desired site, i.e., tumor microenvironment, nanocarriers with cationic surface charge are recovered. Such a switch in surface charge can induce charge disturbance in the tumor microenvironment, which could, to some extent, trigger an adverse impact on the anionic cellular membrane [125, 126], the physiological polyanions (i.e., enzymes, cell receptors) [127], and possibly on the physiological functions of organs. Moreover, after charge reversal, the regenerated positively charged nanocarriers are expected to have a toxic effect, as typical cationic nanocarriers do. Unfortunately, to the best of our knowledge, there is no such study about the potential toxic effect or 43
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the immune response of charge-reversal nanocarriers after the negative-to-positive charge reversal takes place. In view of the critical importance associated with these biosafety issues, it is desirable that these biosafety issues should be thoroughly investigated in future studies.
Summary and outlook
f
7.
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Over the past decade or so, nanocarriers possessing charge-reversal property have
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received increasing attention in the research and development of cancer nanomedicine.
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The realization of charge reversal within a nanocarrier has demonstrated to play a
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crucial role in concurrently achieving prolonged circulation time in the bloodstream and enhanced cellular internalization into cancer ce lls toward targeted release of
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payloads to the intracellular compartments. Biological stimuli, including pH, redox,
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ROS or enzyme, can be readily harnessed to trigger charge reversion to confer
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nanocarriers favorable propensities toward smart cancer nanomedicine. Specifically, charge reversal taking place on the nanocarrier surface can effectively overcome certain benchmark biological barriers in the course of nanocarrier’s systemic transport and intracellular delivery, mainly including reticulo-endothelial system, cellular internalization, and endo/lysosomal membrane disruption. Such strategy is advantageous in that it is essential in enhancing therapeutic efficacy. Despite the aforementioned advantages, one should be aware of the disadvantages associated with charge-reversal nanocarriers. It is worth mentioning that for charge reversal triggered by pH, a precise control of pH-dependent cleavage or hydrolysis 44
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within a narrow pH range is not easy, due to the complex nature of the biological or physiological environment. In addition, upon negative-to-positive charge reversal, the regeneration of the temporarily masked positive surface charge imparts the original core polymer or nanostructure cationic feature. Thus, drawbacks associated with cationic nanocarriers for systemic delivery may “relapse”. One would need to take
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development of practical cancer nanomedicine.
f
these factors into consideration when employing a charge-reversal strategy in the
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As far as diagnostic or theranostic nanocarriers are concerned, several critical
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features could be shared with the therapeutic counterparts to achieve maximum
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efficacies. Thus, the design principle for therapeutic nanocarriers would be valid in such context. The charge-reversal strategy has also been successfully applied to the
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fabrication of smart nanocarriers for cancer diagnostics or theranostics, which shows
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promising potential in improving efficacies. With the advancement in the precise
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synthesis of smart nanocarriers, it is necessary and viable to perform detailed investigations on how charge reversal, taking place in response to varying acidity of tumor extracellular or intracellular microenvironment, would be temporally influenced by difference in charge-reversal moiety. To this end, it is important to gain insights into how fast the chemical structure of pH- labile moieties modulates the charge reversal of resulting nanocarriers. Besides, owing to increasing understanding of cancer biology and with the advancement in nano-biotechnology, it can be envisioned that charge-reversal nanocarriers would continue to be an important arena in the research field of cancer nanomedicine and thus deserves thorough explorations 45
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toward their full potential as precision nanomedicine for cancer diagnosis and treatment. In view of the extensive merits observed in the laboratory research for charge-reversal nanocarriers versus their traditional counterparts, it would be reasonable for one to anticipate improved benefits for their potential applications in clinic settings. In this sense, one may be cautiously optimistic about the clinical
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applicability and benefits of charge-reversal cancer nanomedicine.
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Declarations of interest: none.
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Acknowledgements
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This work was supported in part by the National Natural Science Foundation of China (grant numbers: 51173035, 31670922 and 81771636), the Major Project of
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Wenzhou Science & Technology Bureau (grant numbers ZS2017012, Y20160069),
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and a grant from the 2nd Affiliated Hospital of Wenzhou Medical University.
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58
Journal Pre-proof Table and Figures
Table 1 Stimulus
Site of action for charge reversal
Ref.
Protein
pH
IC
25-28
pDNA Drug
pH pH
IC IC
29-30 35, 53
Drug Drug
pH pH
IC and TME TME
51 52
Drug Drug
pH pH
Drug
Nanogels
Drug
Hybrid micelles
siRNA
Gold NPs Upconversion NPs
siRNA PS
MSNs
Drug
Liposomes Positive-toNegative
Polymer micelles
IC IC，TME
36 50
Enzyme
TME
62
pH
TME
37
pH
IC
80-83
pH pH
TME TME
96 106
pH
TME
113
Drug
pH
TME
124, 125
Drug
ROS
IC
70
Drug
Enzyme
IC
76
oo
Positive
Polymer-drug conjugates
al
Negative-to-
pr
Polymer micelles
f
Payload
e-
Nanocarrier composition
Pr
Type of charge reversal
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rn
IC: Intracellular compartments TME: Extracellular tumor microenvironment PS: Photosensitizer MSNs: Mesoporous silica nanoparticles
59
Pr
e-
pr
oo
f
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Figure 1
60
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(a)
pr
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f
(b)
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Pr
e-
Figure 2
61
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pr
oo
f
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rn
al
Pr
Figure 3
62
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al
Pr
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pr
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f
Figure 4
63
al
Pr
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pr
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f
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rn
Figure 5
64
Pr
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pr
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f
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Figure 6
65
Figure 7
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Pr
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pr
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f
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66
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Pr
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Figure 8
67
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Pr
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pr
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f
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rn
Figure 9
68
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Pr
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pr
Figure 10
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f
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69
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Pr
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Figure 11
70
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Pr
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71
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Pr
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rn
Figure 1
72
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(a)
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(b)
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Pr
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Figure 2
73
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Pr
Figure 3
74
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Pr
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pr
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Figure 4
75
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Pr
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Figure 5
76
Pr
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Figure 6
77
Figure 7
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Figure 8
79
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Figure 9
80
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Pr
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Figure 10
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81
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Pr
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Figure 11
82
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Payload
Stimulus
Site of action for charge reversal
Ref.
Protein
pH
IC
25-28
pDNA
pH
IC
29-30
Drug
pH
IC
35, 53
Drug
pH
IC and TME
51
Drug Drug
pH pH
TME IC
52 36
conjugates
Drug Drug
pH Enzyme
IC，TME
50 62
Nanogels Hybrid micelles
Drug siRNA
pH pH
TME IC
37 80-83
Gold NPs
siRNA
pH
TME
96
Upconversion NPs
PS
pH
TME
106
MSNs
Drug
pH
TME
113
Liposomes
Drug Drug
pH ROS
TME IC
124, 125 70
Enzyme
IC
76
Positive-toNegative
Polymer-drug
Polymer micelles
Drug
Pr
Negative-toPositive
IC: Intracellular compartments
TME
oo
Polymer micelles
pr
Nanocarrier composition
e-
Type of charge reversal
f
Table 1
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TME: Extracellular tumor microenvironment PS: Photosensitizer
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Highlights
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MSNs: Mesoporous silica nanoparticles
Different strategies for charge-reversal cancer nanomedicine are discussed.
Stealth and extended circulation properties can be achieved in systemic applications. Charge reversal at tumor extracellular environment leads to enhanced cellular uptake.
Charge reversal is conducive to enhanced intracellular delivery of therapeutics.
83
Mingzhen Zhang, Xiaoxiao Chen, Chao Li, Xian Shen PII:
S0168-3659(19)30743-6
DOI:
https://doi.org/10.1016/j.jconrel.2019.12.024
Reference:
COREL 10067
To appear in:
Journal of Controlled Release
Received date:
17 October 2019
Revised date:
13 December 2019
Accepted date:
14 December 2019
Please cite this article as: M. Zhang, X. Chen, C. Li, et al., Charge-reversal nanocarriers: An emerging paradigm for smart cancer nanomedicine, Journal of Controlled Release (2019), https://doi.org/10.1016/j.jconrel.2019.12.024
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© 2019 Published by Elsevier.
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Charge-reversal nanocarriers: an emerging paradigm for smart cancer nanomedicine
Mingzhen Zhanga,c# *, Xiaoxiao Chenb# , Chao Lia, Xian Shena,c*
The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou
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a.
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Medical University, 109 Xueyuan West Road, Wenzhou, Zhejiang 325027, P.R.
The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang
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b.
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China
c.
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325000, P.R. China
WMU-Monash University BDI Alliance in Clinical & Experimental Biomedicine,
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Wenzhou Medical University, Wenzhou, Zhejiang 325027, P.R. China
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*Corresponding authors
Email: [email protected] (M.Z.); [email protected] (X.S.)
# These authors contributed equally to this paper.
1
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Abstract
The surface charge of nanoparticles (NPs) plays an essential role in determining their biological properties both in vitro and in vivo. In view of the complex features associated with the biological or physiological microenvironment of solid tumors,
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such as electrostatic interactions between NPs and serum components, cellular
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membrane, or intracellular organelles, drug- loaded NPs (or nanocarriers) should
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intelligently accommodate such unique extra- or intracellular microenvironment in
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order to achieve maximum therapeutic and/or diagnostic efficacy. To that end, the
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surface charge of nanocarriers needs to be readily converted at the target site by means of charge reversal, i.e., conversion from anionic to cationic, or vice versa,
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depending on specific microenvironment. In such a manner, the payloads could be
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efficiently released at the desired tumor site. This review discusses 1) the
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physicochemical aspects related to long-circulating nanocarriers for systemic applications; 2) the recent progress in charge-reversal nanocarriers, which are loaded with drugs, nucleic acids, proteins or imaging agents and triggered by various biological signals (i.e., pH, redox, ROS or enzyme) associated with tumor microenvironment, with an emphasis on those induced by acidic tumoral pH; and 3) the perspectives of charge-reversal nanocarriers regarding thorough investigations on how the chemical structure of charge-reversal moiety temporally affects the responsiveness of the resulting nanocarriers toward the rational design of precision cancer nanomedicine. 2
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Keywords : polymeric micelles; drug delivery; gene delivery; nanomedicine;
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charge-conversional polymers; block copolymer micelles
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1. Introduction Over the past three decades or so, numerous drug delivery systems have been developed and are under intensive investigations as cancer nanomedicine, several of which have been approved for clinical applications while many others are still in different stages of clinical trials. Unfortunately, the overall clinical outcomes of
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cancer nanomedicine is not satisfactory in terms of response rates and survival times
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[1, 2], which has triggered reviews regarding nanoparticle (NP)-based cancer therapy
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Such failures could be associated with the significant discrepancy in the amount of
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NPs between at the site of administration and at the site of action [3].
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The advantage of stealthy drug nanocarriers, realized by the incorporation of a poly(ethylene glycol) (PEG) shell on the nanocarrier surface, has been fully validated
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over the past few decades. PEG is neutral, biocompatible and non- immunogenic and
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has been approved by FDA for clinical applications. In particular, PEG has been
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extensively used for constructing cancer nanomedicine [4-6]. Due to the presence of a PEG shell layer on the NP surface, it has been evidenced that PEG was greatly beneficial for prolonged circulation time and enhanced accumulation at tumor site, owing to the EPR effect [6, 7]. However, it has also noticed that the neutral PEG layer was not optimal for cellular uptake given that cell membrane is negatively charged. Therefore, it has been conceived that an ideal nanocarrier should concurrently exhibit the following favorable features: 1) long circulation time and minimal clearance by the reticulo-endothelial system (RES) while in the blood stream; 2) efficient cellular uptake by tumor cells upon arrival at the tumor milieu; and 3) efficient release of 4
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payloads at the targeted intracellular compartment. The emergence of precision medicine for cancer therapy and/or diagnosis can greatly help address the prior challenges encountered for polymeric cancer nanomedicine. With the recent advances in polymer chemistry and by leveraging a wealth of knowledge in tumor biology, it is highly desirable to develop new strategies
f
toward intelligent drug nanocarriers (NCs) by which nanocarriers can exhibit
oo
favorable physicochemical features to accommodate different physiological or
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biological environments, either in the bloodstream, at the tumor milieu, or inside the
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tumor cells, to maximize their biological functions, and eventually therapeutic
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outcomes. The difference in certain biological signals (i.e., pH, redox, ROS and enzyme) existing between the above- mentioned biological microenvironments can be
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utilized as a triggering stimulus toward smart drug nanocarriers, which can respond to
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the stimulus in a site-specific manner and thus exhibit promising propensities toward
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cancer nanomedicine with a potential for clinical applications. Owing to the recent advances in the physicochemical characterization of NPs, it has been demonstrated that the intrinsic features of NPs, such as size, shape and surface charge, play a critical role in determining their biological functions both in vitro and in vivo [8-16]. Furthermore, increasing understanding about tumor biology, in particular, about the tumor microenvironment (TME), could allow for thorough elucidation of the relationship between the physicochemical properties and the biological properties of NPs. In this review, we will focus on how the surface charge of NP (or nanocarrier) interplays with diverse physiological or biological 5
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microenvironment of solid tumors (i.e., intra- or extracellular microenvironment) and how the surface charge of NP plays an essential role in directing the rational design of therapeutic and/or diagnostic agent- loaded NPs toward personalized cancer nanomedicine. First, we provide an overview about the relevant physicochemical foundations of long circulating nanocarriers for systemic delivery of therapeutic
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and/or diagnostic agents, followed by a discussion of different strategies for
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fabricating smart charge-reversal nanocarriers responsive to unique biological signals,
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including pH, redox, ROS and enzyme, in the tumor intra- or extracellular
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microenvironment. The charge-reversal strategy is advantageous for enhanced
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therapeutic and/or diagnostic efficacy and thus may provide a useful solution to
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facilitate the clinical translation of relevant cancer nanomedicine in the future.
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2. Charge-reversal nanocarriers: a powerful solution to address the polycation
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dilemma in the systemic applications of NPs In order to fully understand how the surface charge of NP plays a key role during its journey from the site of administration to the site of action, it is of great importance to have a basic understanding about how the surface charge influences the bio- function of NP in the context of systemic drug delivery, thus providing a rationale for the construction of smart NP by utilizing a novel concept, namely charge reversal. It has been demonstrated that NPs with positive surface charge show significantly higher affinity with negatively charged cell membranes, which is essential for enhanced membrane penetration and cellular uptake [17-20]. Stellacci et al. provided 6
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a valuable summary about how the surface properties (i.e., size, surface charge and surface ligand) of NPs influence the interactions between NPs and cells [17]. By providing insights into NPs of various surface properties, efforts in mechanismic elucidation were actively pursued. It was concluded that surface properties of NPs play a critical role in modulating their interactions with cells. Chen et al. showed that
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more positively charged hydroxyapatite (HAp) NPs could readily penetrate into
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osteoblast cells compared with negatively charged HAp NPs with similar size and
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shape, which was attributed to the attractive or repulsive interaction between cell
e-
membranes and different charged NPs [18]. Mukherjee et al. studied how gold NPs
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(AuNPs) of various surface charges interacted with plasma membrane of benign or malignant cells [19]. They found that the surface charge of AuNPs plays a
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intracellular events.
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determining role in modulating membrane potential and subsequent downstream
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On the other hand, positively charged NPs can induce significant side effects, in particular in systemic applications, i.e., strong interactions with serum components in the blood and non-specific cytotoxicity against normal cells [19, 21], resulting in rapid clearance, hemolytic side effect and toxicity to normal tissues [22, 23]. In this sense, positively charged nanocarriers are not suitable for in vivo applications. Thus, the development of charge reversal or zwitterionic NPs, which display neutral or negatively charged in the bloodstream but reverse to positively charged upon at the tumor microenvironment, are highly desirable for the systemic delivery of NPs loaded with therapeutic and/or diagnostic agent to cancerous tissues. Due to the site-specific 7
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negative-to-positive charge reversion taking place on the NP surface at the mildly acidic tumor milieu or inside the acidic endo/lysosomal compartments, significantly improved cellular uptake by tumor cells or efficient endosomal escape would be realized while the long circulating, minimal non-specific toxicity features of stealthy NPs are preserved in the negatively charged status while in the bloodstream [24].
Nanocarrier
reversal
composition
Payload
25-28
pH
IC
29-30
pH
IC
35, 53
Drug
pH
IC and TME
51
Drug
pH
TME
52
Drug
pH
36
Pr
Drug
pH
Drug Drug
Enzyme pH
TME TME
62 37
Hybrid micelles
siRNA
pH
IC
80-83
Gold NPs
siRNA
pH
TME
96
Upconversion NPs
PS
pH
TME
106
MSNs
Drug
pH
TME
113
Liposomes
Drug
pH
TME
124, 125
Polymer micelles
Drug Drug
ROS Enzyme
IC IC
70 76
Nanogels
Positive-toNegative
al Drug
IC IC，TME
Polymer-drug conjugates
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Positive
charge reversal
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Negative-to-
Ref.
IC
pDNA Polymer micelles
Site of action for
pH
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Protein
Stimulus
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Type of charge
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f
Table 1. Representative examples of charge-reversal nanocarriers with different compositions and triggering mechanisms
50
IC: Intracellular compartments TME: Extracellular tumor microenvironment PS: Photosensitizer MSNs: Mesoporous silica nanoparticles
3. Charge reversal from anionic to cationic As mentioned previously, the presence of various site-specific biological stimuli 8
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(i.e., pH, redox, ROS, enzyme or a combination of two) could be utilized to trigger charge reversal of NPs. Among these stimuli, a pH gradient between the physiological and the extracellular environment of solid tumors is one of the most frequently used triggering signals. The pH value of the physiological environment is around 7.4 whereas the extracellular environment of solid tumors typically features a pH value
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between 6.4-6.8. Furthermore, endo/lysosomal compartments display a more acidic
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pH value, i.e., 4.5-5.5. The presence of such pH gradient between the extracellular
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and intracellular environments of solid tumors indeed endows a unique opportunity
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for the precision design of smart drug delivery systems targeting the tumor site.
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Therefore, by taking advantage of such site-specific pH values, it is feasible to develop pH-responsive drug delivery systems which can effectively circ umvent biological
barriers
of
the
tumor
extra-
and/or
intra-cellular
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complex
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microenvironment and intelligently release payloads to the targeted locations inside
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the cancer cells to maximize therapeutic outcome. In a pH-induced negative-to-positive charge-reversal strategy, nanocarriers composed of or decorated with a biocompatible polymer possessing pH-dependent charge-reversal moieties are fabricated. These moieties exhibit negatively charged at physiological pH while becoming positively charged at acidic pH, either in the extracellular microenvironment of solid tumors or inside the acidic endo/lysosomal compartments. 3.1.Charge reversal triggered by pH Typically, negative-to-positive charge reversion can be realized through the 9
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pH-dependent transformation of amides into amines occurring on a polymer with charge-reversal moieties. For instance, nanocarriers prepared with a charge-reversal polymer exhibit net negative surface charge in the blood stream but undergo negative-to-positive conversion at the tumor site. In some cases, such nanocarriers further display membrane disruption capability when inside the acidic endo/lysosomal
Charge reversal triggered by acidic endosomal/lysosomal compartments
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3.1.1.
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compartments, owing to a proton sponge effect.
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Kataoka and coworkers reported pioneering investigations in which they used
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different maleic anhydride (MA) derivatives to modify the end group of a protein (i.e.,
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equine heart cytochrome c, CytC) or the side chain of homo- or copolymer of polypeptide, such as polyaspartate (PAsp), PEG-polyaspartate (PEG-PAsp) or
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polylysine to construct pH- labile charge-reversal polymeric micelles for the
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intracellular delivery of proteins [25-28] and pDNA [29, 30]. In order to address the
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challenges encountered in protein delivery, i.e., insufficient charge density of protein to form stable protein/polymer complexes, in one study, they first modified the lysine units of a model protein, CytC, with maleic anhydride (MA), i.e., citraconic anhydride or cis-aconitic anhydride, to respectively produce citraconic amide or cis-aconitic amide and to reversibly increase the anionic charge density of the precursor CytC [26]. It was found that by selecting the number of carboxyl group on the MA, i.e., citraconic anhydride (with 2 COOH) or cis-aconitic anhydride (with 3 COOH), the charge density of the MA- modified CytC was increased in accordance with the number of COOH groups. The charge density of the modified CytC was respectively 10
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increased to -510 Da and -320 Da per charge for citraconic acid and cis-aconitic acid, which is significantly enhanced when compared to the unmodified CytC (i.e., +1391 Da per charge). The resulting anionic CytC was then complexed with a cationic polymer,
namely,
PEG-poly{N-[N’-(2-aminoethyl)-2-aminoethyl]aspartamide}
(PEG-PAsp(DET)) copolymers, to form polyion complex (PIC) micelles. Upon
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internalization into cells and being transported inside the acidic endo/lysosomes,
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maleic amides on the CytC underwent pH-dependent cleavage to expose primary
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amines, thus achieving charge reversal from negative to positive. Thus, charge
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repulsion took place between CytC and the DET-containing polymer, both bearing
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positive charges. Consequently, CytC was expelled from the PIC micelles and released into cytoplasm.
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Moreover, instead of decorating proteins with charge reversal moieties, Kataoka
rn
also used charge-reversal MA derivatives (i.e., cis-aconitic and citraconic anhydride)
Jo u
to modify a polycation, i.e., PAsp(DET), in order to achieve net negative charge. The resulting polymer was used to complex with positively charged lysozyme to form PIC micelles. Likewise, the aconitic amide moiety within the PIC micelles underwent negative-to-positive conversion, which led to the repulsion of lysozyme from the PIC micelles and released to cytoplasm [27].
11
pr
oo
f
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e-
Figure 1. Preparation of charge-reversal PIC micelles containing CytC derivatives and PEG–PAsp(DET). a) Citraconic anhydride (or cis-aconitic anhydride/succinic anhydride). Reproduced with permission from Ref. [26]. Copyright 2009, John Wiley
Pr
& Sons.
al
In addition to their applications in protein delivery, charge-reversal PEG-based
rn
copolymers were also widely used to construct non- viral vectors for pDNA delivery
Jo u
[29]. It was found that for polycation-based non-viral gene delivery systems, increasing amine density of the polycation or a higher N/P ratio is favorable for membrane destabilization but it induces adverse effect (i.e., cytotoxicity and immune response) due to non-specific interaction with negatively charged serum proteins in the blood. A charge reversal polymer has demonstrated to effectively address the above dilemma. For example, a tertiary system, consisting of pDNA, pAsp(DET) and a charge-reversal polymer pAsp(DET-Aco), was used to delivery pDNA to cells. Poly(aspartic acid) having DET as a flanking group on the side chain (PAsp(DET)) is a cationic polymer and displays endosomal disrupting capability conferred by the 12
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DET moiety. When it was modified with a MA derivative, i.e., aconitic anhydride (Aco), pAsp(DET-Aco) was obtained. As discussed above, aconitic amide undergoes pH-dependent cleavage while at acidic endo/lysosomal compartments and results in unleashing of DET groups, which subsequently exerts endosomal membrane disruption and facilitates PIC micelles to escape from the endosomes. On the other
f
hand, due to the negative-to-positive charge conversion taking place for the Aco
oo
moiety, the cationic PAsp(DET) repels the positively charged pAsp(DET)/pDNA
pr
polyplex NPs. More importantly, owing to the charge reversal- induced synergic effect,
e-
efficient endosomal escape was observed for the polyplex NPs, presumably benefited
Pr
from the proton sponge effect conferred by the DET moiety. It is evidenced that as pH value was gradually decreased from neutral to acidic,
al
the DET group underwent two-step protonation and exhibited proton sponge effect,
rn
facilitating NC escaping from the late endosomes or lysosomes. For example, when
Jo u
the DET end group of PEG-PAsp(DET) is reacted with citraconic anhydride, it is changed to citraconic amide. Quantification of amine concentration was performed, which shows that the amide group degraded in the acetate buffer (pH 5.5) within 1 h while 60% of the amide groups remained intact at pH 7.4 during a period of 5 h. In contrast, no degradation occurred for the negative control, where a non-pH- labile succinic anhydride was used to react with DET.
13
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(a)
oo
f
(b)
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Figure 2. (a) Charge-reversal ternary polyplexes with endosome-disrupting function; (b) Synthesis of PEG-PAsp(EDA-Cit). Reproduced with permission from Ref. [29] and Ref. [27], respectively. Copyright 2008, John Wiley & Sons; Copyright 2007,
Pr
e-
American Chemical Society.
In the context of intracellular delivery of anti-cancer drugs using NPs, the
al
anti-cancer drug, such as camptothecin (CPT), doxorubicin (Dox) and cisplatin, needs
rn
to interact with DNA located in the cell nuclei to induce cell apoptosis [31-34]. In
Jo u
order for the drug to diffuse into the nuclei, it also needs to overcome multiple barriers, which requires that the drug-loaded NPs adapt themselves to the changing biological environments during their journey to the cell nuclei. Shen
and
coworkers
prepared
pH-dependent
polymeric
micelles
and
polymer-drug conjugates for nuclear drug delivery [35, 36] by utilizing a charge-reversal strategy.
In one
study,
polycaprolactone-b-polyethyleneimine
(PCL-b-PEI) copolymer was synthesized, in which part of the primary and secondary amine groups of PEI were masked with amides by using 1,2-cyclohexanedicarboxylic anhydride, i.e., PCL-b-PEI/amide [35]. It was found that majority of these amide 14
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groups promptly hydrolyzed at pH 5-6, while only 50% hydrolyzed at pH 7.4 after 60 h. Such observation was confirmed by zeta potential measurements, in which the resulting PCL-b-PEI/amide micelles maintained a zeta potential of ~ -20 mV at pH 7.4 over a period of over 60 h while the value gradually increased to be slightly positive at pH 6, and further reached +50 mV at pH 5, clearly demonstrating the
f
occurrence of a negative-to-positive conversion. As shown in the cellular
oo
internalization using flow cytometry, the Dox- loaded charge-reversal NPs showed
pr
faster cellular internalization at pH 6 than at pH 7.4, presumably due to the
e-
regeneration of positive charges at pH 6 which consequently promoted the cellular
Pr
internalization via electrostatically adsorptive endocytosis. In addition, intracellular trafficking and nuclear localization of Dox- loaded charge reversal NPs was performed
al
on SKOV-3 cells using confocal laser scanning microscopy (CLSM), which
rn
demonstrated that charge reversal improved affinity with the nuclear membrane at 8 h
Jo u
post-incubation, followed by NPs entry into the nuclei of cancer cells. Consequently, significant cytotoxicity was observed, as evidenced by dramatically decreased IC 50 . In another study, a polymer-drug conjugate, consisting of poly(L- lysine) (PLL) and camptothecin (CPT), was fabricated for the nuclear delivery to cancer cells [36]. In order to minimize the cytotoxicity induced by the cationic PLL while enhancing cellular uptake efficiency, the following strategy was employed. First, part of the PLL side chain was modified using 1,2-dicarboxylic cyclohexene anhydride (DCA) to form pH-sensitive amides and thus to mask the positive charge. The PLL chain could be regenerated upon at the acidic extracellular microenvironment of tumor. Second, 15
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CPT was conjugated to PLL using a redox-sensitive linker (i.e., disulfide bond), which is responsive to the significantly augmented glutathione concentration in the intracellular environment but remains stable in the extracellular environment. Third, a folic acid (FA) ligand was introduced to PLL to achieve enhanced cellular uptake on FA-overexpressing cancer cell lines. The results show that the negative-to-positive
f
charge reversal induced significant enhancement in the cytoxocity of CPT, likely due
oo
to increased dose of CPT released in the nucleus, as compared to the case in which
Jo u
rn
al
Pr
e-
pr
drug resistance was typically the observed for CPT.
Figure 3. The structure of targeted charge-reversal PLL conjugate and tumor acidity-triggered charge reversal (a) and nuclear drug delivery (b). Reproduced with permission from Ref. [36]. Copyright 2009, John Wiley & Sons.
3.1.2.
Charge reversal induced by mildly acidic extracellular microenvironment of
solid tumors
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The vicinity of solid tumors is considered as a first line of barriers for NPs to enter tumor tissues. It has been found that the tumor extracellular microenvironment displays mild acidity, i.e, acidic pHe, compared to the neutral conditions in the bloodstream. Therefore, such pH difference could be utilized as a useful stimulus for charge reversion.
f
Wang and coworkers proposed to mask the terminal amines of poly(2-aminoethyl
oo
methacrylate hydrochloride) (PAMA) with 2,3-dimethylmaleic anhydride (DMA)
pr
such that amines were converted into amides. The resulting polymer was used to
e-
fabricate pHe-triggered charge-reversal nanogels [37], of which the amide bond was
Pr
stable at neutral and alkali pH values, but hydrolyzed promptly in response to acidic tumor pHe and recovered the positive-charged amines in the negatively charged
al
PAMA-DMA nanogel. Such charge reversion was verified by zeta potential and 1 H
rn
NMR measurements. As a result of charge reversion at pH 6.8, the zeta potential
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increased significantly, from negative to neutral, within a short period of time (i.e., 35 min) and continued to increase for a prolonged time. In contrast, the zeta potential increased much more slowly at pH 7.4. In the case of succinic anhydride- modified PAMA nanogel, which is a non-charge reversal control, no charge reversion was observed. Furthermore, CLSM and flow cytometry were employed to investigate the cellular internalization of nanogel at pH 7.4 and 6.8, which led to significantly different observations. Remarkable internalization was observed for the nanogel at pH 6.8, as evidence by dramatic fluorescence in the cytoplasm. Howeve r, the nanogel was primarily stick to the cell membrane at pH 7.4. The difference can be explained 17
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by the fact that the negative-to-positive conversion enhances the interactions between the positively charged nanogel and the cell membrane, leading to enhanced cellular internalization. The impact of charge reversal was further corroborated with protein absorption with BSA, in which minimal protein absorption was observed at pH 7.4 while strong absorption occurred at pH 6.8 for the charge-reversal nanogel.
f
For the Dox- loaded nanogel, a significantly different drug release trend was
oo
observed. Charge reversion resulted in accelerated Dox release, dependent on acidity,
pr
due to decreased interaction between the positively charged Dox and the
e-
PAMA-DMA nanogel with increasingly protonated COOH groups and the resultant
Pr
positive charges resulting from charge reversal. Overall, Dox- loaded charge-reversal
al
nanogels exhibited significantly improved cytotoxicity.
rn
3.1.3. Charge reversal induced by stepwise response to increasing acidity from
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extracellular tumor microenvironment to endo/lysosomal compartments In addition to biological barriers encountered during the course of intracellular delivery, multidrug resistance (MDR) has also been widely recognized as one of the most challenging and common barriers in the context of cancer chemotherapy due to efflux
of chemotherapeutic
P-glycoproteins (Pgp),
drugs
from cytoplasm by the overexpressed
leading to dramatically reduced therapeutic efficacy [32, 38,
39]. Therefore, bypassing the MDR mechanism would be a critically important for the nuclear delivery of anticancer drugs. One useful strategy to address this challenge is to introduce nuclear localization signal (NLS) to drug delivery systems, such as a TAT 18
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peptide, a cationic peptide derived from human immunodeficiency virus (HIV) [40]. TAT peptide has found extensive applications in transporting various cargos from cytosol to nuclei [41-45], i.e., nucleic acids [41-43], proteins [44-46] and nanoparticulate delivery systems [47-49], in that it can be recognized by nuclear pore complexes (NPC). Therefore, TAT peptide should be helpful in circumventing the
f
Pgp-associated MDR pathway.
oo
Drug delivery systems, consisting of charge-reversal polymers or based on
pr
polymer-drug conjugates, have demonstrated superior propensities as intracellular
e-
drug delivery systems to solid tumors. In general, the negative-to-positive charge
Pr
reversal, realized by masking the positive charges on the drug carrier surface using a maleic anhydride derivative, takes place at the mildly acidic extracellular
al
microenvironment of solid tumors. Such strategy has demonstrated to be useful in
rn
addressing the dilemma associated with long circulation time and minimal
carboxylic
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non-specific interactions between drug carriers and serum component. When the group
underwent pH-dependent
hydrolysis
in
the extracellular
microenvironment, the positive charges were readily regenerated, which resulted in remarkably enhanced cellular uptake and cytotoxic ity on cancer cells. It was also recognized that upon internalization via endocytosis, these single pH-responsive drug delivery systems need to overcome additional pH-associated biological barrier located inside organelles, namely, endo/lysosomes, in order to readily release drug payload. As mentioned above, the pH value inside the endo/lysosome compartments is 4.5-5.5, which is significantly lower than that in the extracellular microenvironment and thus 19
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requires a second mechanism by which the linker between the drug and the polymer undergoes prompt cleavage at endo/lysosomal pH to facilitate drug release. To concurrently achieve the above goals, Wang and coworkers further prepared a dual pH-responsive polymer-drug conjugate, consisting of polyphosphoester (PPC) and Dox [50]. The polymer was first conjugated with Dox through a hydrazone (Hyd)
f
linker and was then end-modified with a maleic anhydride derivative (i.e., DMA) to
oo
produce amide bonds. The resulting hydrazone and maleic amide linkages are acid
pr
labile and can respond to mildly acidic and endo/lysosomal pH value, respectively.
e-
The pH-dependent cleavage of maleic acid amide and hydrazone linkage was
Cellular
internalization
and
Pr
confirmed by 1 H NMR and the release of Dox at desired pH range, respectively. intracellular
trafficking
were
performed
on
al
MDA-MB-231 cells using flow cytometry and CLSM, respectively, which showed
rn
enhanced uptake of PPC-Hyd-Dox-DA NPs and efficient release of Dox from the NP
Jo u
release, and improved presence in the cell nuclei, as compared to controls. These results are attributed to the dual responsiveness toward a pH gradient, i.e., pHe 6.5 for the extracellular tumor microenvironment and pH 4.5-5.5 for acidic endo-/lysosomes. Moreover, the dual pH-responsive propensity exhibited great potential in inhibiting cancer stem cells, clearly demonstrating the advantage of a dual pH-responsive drug NC.
20
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As mentioned previously, bypassing the Pgp-associated multidrug resistance (MDR) is critical to achieve enhanced therapeutic efficacy in cancer chemotherapy using a drug delivery system. Given that the cationic TAT peptide, a type of cell
f
penetrating peptides (CPP), has shown to be helpful in circumventing the
oo
Pgp-associated MDR mechanism, the introduction of TAT peptide to a charge-reversal
a
polypeptide,
namely
Poly(L- lysine)-block–poly(L- leucine)
e-
synthesized
pr
drug delivery system could be advantageous. To this end, Zhang and coworkers
Pr
(PLL-b-PLLeu) [51]. The lysine residue was amidized with dimethylmaleic anhydride (DMA) derivative to construct negatively charged micelles, possessing nuclear
al
localization and charge reversal properties. Moreover, the TAT peptide was amidized
rn
with succinyl chloride (SA), an acid-degradable moiety, to mask its positive charge
Jo u
and cell-penetrating features and was then conjugated with the polypeptide. The resulting polypeptide, modified with two acid- labile moieties responsive to two different pHs, was used to fabricate Dox-loaded PLLeu-PLL(DMA)-TAT(SA) micelles. In view of the presence of two pH- labile moieties within the same micelles, the micelles underwent sequential charge reversal, first for the DMA moiety at mildly acidic microenvironment and then for the SA moiety in the endo/lysosomal compartments. Consequently, in addition to TAT being regenerated to confer cell-penetrating capability, micelles also became positively charged, which led to significantly increased delivery of Dox to the cell nuclei, as evidenced from the flow 21
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cytometry measurement and CLSM observation. Thus, the notion of dual pH-responsive polymers or polymer-drug conjugates has clearly demonstrated the advantages of realizing dual negative-to-positive charge reversals at two pH values within the same drug carrier, first in the tumor microenvironment (pH 6.8) and then in the endo/lysosomal compartments (pH
f
4.5-5.5). Such strategy not only extends circulation time and minimizes non-specific
oo
interaction with serum components, it also facilitates the nuclear delivery of
pr
anticancer drug, as demonstrated by significantly increased cytotoxicity to cancer
Jo u
rn
al
Pr
e-
cells.
Figure 5. Schematic illustration of (A) the self-assembled polypeptide micelles stepwise responding to mildly acidic tumor tissues through a charge reversal process and to more acidic endosomes to reactivate the nuclear location function of TAT peptide; (B) cellular uptake of nanoparticles and nuclear delivery of drugs. Reproduced with permission from Ref. [51]. Copyright 2015, John Wiley & Sons.
3.1.4
Charge reversal as a means to achieve size-shrinkable nanoparticles 22
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It has been revealed that size of NPs plays a critical role in the biological property in the context of polymeric cancer nanomedicine. Kataoka et al. demonstrated that the size of polymeric micelles strongly affects the biological properties, such as biodistribution and tumor penetration in xenograft pancreatic tumor model [8]. The results showed that although NPs having a size in the sub-100 nm range is optimal to
f
achieve long blood circulation and enhanced tumor retention via the EPR effect,
oo
smaller size is critical for deep tumor penetration, thus enhanced antitumor efficacy.
pr
In order for chemotherapeutic agents (such as Dox, CPT and cisplatin) to exert
e-
biological functions (i.e., inducing apoptosis), such agents must enter into cell nucleus
Pr
and interact with DNA molecules located there. In addition to the aid of NLS, NPs of small size become an essential factor in determining whether they could efficiently
al
enter the nucleus. In other words, small-sized NPs are required. In order to
rn
concurrently address the conflicting size requirements required for the EPR effect (i.e.,
Jo u
sub-100 nm) and nuclear entry (i.e., sub-50 nm), an ideal strategy would be to design a nanocarrier which exhibits sequential size shrinking, depending on the stage of particle transportation or on the particle location, i.e., from blood circulation to location in the cytoplasm or inside the endo/lysosomes. In accordance with such a notion, a NC system should contain two different types of functional gro ups in order to achieve two-stage size shrinking, taking place respectively in the extracellular and intracellular environment. Shen
and
coworkers
used
a
maleic
anhydride
derivative
(i.e.,
1,2-dicarboxylic-cyclohexene anhydride, DCA) to mask majority of the positively 23
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charged amino termini on PAMAM-G5 to confer negative charges, which is beneficial for prolonged circulation time and minimal non-specific interaction with serum component in the blood [52]. Upon at the acidic tumor microenvironment (TME), hydrolysis of amides led to regeneration of NH2 termini as well as positive charges, thus facilitating cellular uptake by cancer cells. Moreover, the remainder of the
f
terminal NH2 groups was modified with folic acid ligand and thiolated CPT,
oo
respectively. The advantage of thiolated CPT is that when the NC is internalized into
pr
cells, the disulfide linker breaks up and CPT can be released from the NC due to
e-
significantly elevated glutathione (GSH) concentration inside the cells. As a
Pr
consequence of such stepwise cleavage of amide and disulfide linkages occurring respectively at extracellular and intracellular environment, dramatic decrease in
al
particle size was achieved, which resulted in enhanced drug delivery to the nucleus,
rn
clearly demonstrative of the merits of a charge reversal strategy.
Jo u
In another study, Huang et al. prepared N-(2-hydroxypropyl) methacrylamide (HPMA)-based nanovehicles constructed via electrostatic interaction between oppositely charged polymers [53]. The anionic component was obtained by modifying the HPMA side chain with charge reversal moieties, i.e., DMA, while the cationic component contained intracellular detachable subgroup (IDS), consisting of Dox and a cell penetrating peptide (CPP), i.e., octaarginines peptide and an NLS (R8NLS). Under physiological conditions, the pristine nanovehicles exhibited neutral surface charge and suitable particle size, thus possessing propensities for prolonged blood circulation and enhanced tumor accumulation through the EPR effect. Upon at the 24
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acidic TME, or first-stage tumor acidity, charge reversal of the anionic component took place. Consequently, the nanovehicles dissociated and transformed into much smaller conjugates, leading to the exposure of the R8NLS species to facilitate cellular uptake. Next, upon localization in the more acidic endo/lysosomal compartment, the nanospecies underwent a second-stage size reduction wherein the IDS detached from
f
the copolymer and further shrank its size to enhance NLS-mediated nuclear pore
oo
complex transport. By means of FRET and CLSM, two-stage size shrinking led to
pr
remarkably enhancement in cellular uptake and nuclear entry of the Dox-containing
Jo u
rn
al
Pr
e-
species, in consistent with the improved anticancer efficacy in vivo.
Figure 6. Schematic illustration of a multi-stage size reduction strategy using HPMA-based polymeric nanovehicle (PNV). i) Temporary shielding of cationic P-DoxR8NLS using charge-reversal P-DMA, which is anionic at neutral pH to prolong circulation. ii) Self-assembly into PNV through electrostatic attraction, leading to size increase and enhanced EPR effect. iii) Acidic tumor extracellular microenvironment results in charge reversal in P-DMA and collapse of PNV into smaller copolymer chains. iv) Exposure of R8NLS enhances internalization into tumor cells. v) Intracellular release of IDS due to acidic hydrolysis of the hydrazone linkage in the acidic endo/lysosomal compartments, leading to a second-stage size reduction. vi) Endo/lysosomal escape and NLS-assisted nuclear targeting. Reproduced 25
Journal Pre-proof with permission from Ref. [53]. Copyright 2015, John Wiley & Sons.
3.2. Enzyme-triggered charge reversal In addition that positively charged nanocarriers exhibit dramatically enhanced cellular uptake and are beneficial to overcome biological barriers toward improved therapeutic efficacy, it has been recognized that the ATP-dependent transcytosis,
oo
f
which transports cargos across the endothelium, plays a vita l role in enhancing the transport of nanocarriers across the capillary wall into tumor tissues. Bae et al. have
pr
demonstrated that cationic nanocarriers can effectively promote adsorption- mediated
e-
transcytosis (AMT) and facilitates nanocarriers to penetrate across multiple cell layers
Pr
[54]. As mentioned above, cationic nanocarriers are not suitable for in vivo
al
applications since they compromise the long circulation property and induce
rn
significant non-specific toxicity. A charge-reversal strategy could be an effective solution to this challenge, in which NPs display different surface charges during blood
Jo u
circulation or in the vicinity of tumor tissues. As extensively discussed above, a pH gradient has been widely utilized to trigger negative-to-positive charge reversal and thus is able to fulfill that goal when nanocarriers are navigated from the neutral physiological environment to the mildly acidic tumor microenvironment or to the acidic endo/lysosomal compartments. However, in the context of luminal and perivascular compartments, the application of a pH gradient is limited not only by the presence of the local neutral environment [55], but also by the fact that mildly acidic microenvironment is so far away from the tumor blood vessel networks (i.e., 100-200 m) to exert an effective pH gradient [56, 57]. 26
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In order to overcome the challenges associated with the pH-triggered charge reversal taking place in the luminal and perivascular regions, a novel strategy was necessary. It has been found that cell surface enzyme, i.e., γ-glutamyl transpeptidase (GGT), can be used to cleave γ-glutamyl bonds, i.e., -glutamylamides. Considering that GGT is overexpressed on the surface of endothelial cells in various human
f
tumors (i.e., liver, cervical and ovarian cancers) [58, 59], GGT could be utilized as a
oo
potential target in the design strategy of cancer therapy and cancer imaging probes [60,
pr
61]. By means of selective cleavage of γ-glutamyl bonds, i.e., -glutamylamides of
e-
glutathione (GSH), GGT can be used to fulfill certain biological functions associated
Pr
with cancer cells.
Based upon these findings, Shen et al. recently proposed to synthesize a polymer-drug
conjugate,
al
GGT-responsive
acrylamide)
of
(PBEAGA)
and
rn
poly(2-(l-γ-glutamyl- l-α-aminobutyrylamino)ethyl
consisting
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camptothecin (CPT) (PBEAGA-CPT). The conjugate remained negatively charged in the bloodstream to exert prolonged circulation and stealthy properties. Upon contact with the tumor endothelial cells, which features elevated GGT expression level, the conjugate underwent GGT-mediated cationization, namely GGT-catalyzed hydrolysis of -glutamylamide to produce an amino derivative, resulting in negative-to-positive charge reversal to the conjugate [62]. The resulting cationic conjugate was revealed for
efficient
cellular
internalization
and
transcytosis,
allowing
enhanced
transendothelial and transcellular transport. Consequently, improved therapeutic efficacy was expected. As evidenced from the zeta potential measurements, the zeta 27
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potential of the PBEAGA-CPT conjugate changed from negative to positive within 15 h in the presence of 10 U/mL GGT, which was not observed for the control not containing a GGT-sensitive bond. In vitro experiments showed that compared to a control, i.e., GGT-negative fibroblast cells, only the GGT-positive cancer cells displayed significantly increased cytotoxicity. These results were substantiated with
f
cellular internalization pathway and intracellular trafficking studies using 2D cell
oo
culture, as well as with cellular penetration studies using multi-cellular spheroids to
pr
mimic tumor tissues, which demonstrated that the penetration of the GGT-sensitive
e-
PBEAGA-CPT conjugate was through a transcytosis mechanism dependent on the
Pr
caveolae- mediated endocytosis and exocytosis. In vivo experiments revealed the preferential accumulation of PBEAGA-CPT conjugate in the GGT-overexpressed
al
liver tumor site (i.e., HepG2) and dramatically enhanced antitumor efficacy, as
rn
evidenced from the eradicating of large tumors (i.e., subcutaneous HepG2) and extend
Jo u
survival rate of mice bearing orthotopic pancreatic tumors.
28
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Figure 7. Schematic representation and zeta potential characterization of the enzyme-activatable polymer–drug conjugate. Reproduced with permission from Ref. [62]. Copyright 2019, Springer Nature.
4. Charge reversal from cationic to anionic The strategy of stimuli-triggered charge reversion, typically reversion from
oo
f
anionic to cationic, has been widely employed in the rational design of NC as cancer
pr
nanomedicine, and has contributed to significant progress in the development of cancer nanomedicine toward clinical applications. Recently, the concept of
e-
cationic-to-anionic charge reversal has received a great deal of interest in nanocarrier
Pr
design.
al
In the field of non- viral gene vectors, cationic polymer or liposomes have been
rn
extensively used to construct nano-sized polyplexes or lipoplexes to deliver nucleic
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acid (pDNA or siRNA) payload to cells, where the nucleic acid exerts its biological functions. However, one complication associated with cationic vector is that the positive charge, presented on the dissociated cationic debris, was found to interact and interfere with the intracellular DNA transcription machine, thus leading to non-specific toxicity [63, 64]. To address such a drawback, a new paradigm of non-viral gene vector, which does not rely on the conventional electrostatic attraction between a cationic vector and an anionic nucleic acid to form polyplexes, should be useful since the dissociation of polyplexes between the cationic vector and the anionic nucleic acid generates a large
29
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amount of short cationic debris harmful to DNA transcription machine. Instead, a new class of gene vector would depend on the electrostatic repulsion between two anionic species, i.e., nucleic acid and a charge reversal polymer, to deliver payloads. Such strategy should be a promising alternative in that it can eliminate the production of
f
cytotoxic cationic debris, as is the case for the conventional gene vectors.
oo
4.1.ROS-mediated charge reversal from cationic to anionic
pr
It has been demonstrated that, owing to oncogenic transformation, cancer cells
e-
generate higher levels of intracellular reactive oxygen species (ROS), including H2 O 2 ,
Pr
hydroxyl radical, and superoxide, than normal cells [65, 66]. Thus, such feature of cancer cells could be utilized as a stimulus to mediate efficient intracellular delivery
one
study,
Shen
Jo u
In
rn
have been reported [67-72].
al
of payloads using smart nanocarriers. Several ROS-responsive polymeric nanocarriers
poly[(2-acryloyl)ethyl(p-boronic
et
al. acid
synthesized
a
cationic
benzyl)diethylammonium
polymer, bromide]
(B-PDEAEA), which was modified with a reactive oxygen species (ROS)-sensitive charge-reversal moiety capable of positive-to-negative conversion [70]. Upon at elevated ROS (i.e., H2 O2 ) level inside tumor cells, it could oxidize boronic acid groups by ROS, leading to release of p- hydroxylmethylenephenol (HMP) from the quaternary ammonium [73] and resulting in the formation of tertiary amines. Consequently, it catalyzed the conversion from polyacrylate to anionic poly(acrylic acid) [74]. As a result of hydrolysis of ester group to produce carboxylic acid, i.e., 30
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poly(acrylic acid), the zeta potential underwent positive-to-negative conversion in a certain timeframe, depending on pH value. For instance, it accelerated with decreasing pH value. Due to the ROS-induced charge reversal, anionic nucleic acid was readily released from the polyplexes without interfering with the DNA transcription machine, thus dramatically minimizing the non-specific toxicity. As
f
shown in the gene transfection experiments using a reporting gene, the gene vector
oo
prepared with a charge-reversal polymer (i.e., B-PDEAEA) exhibited significantly
pr
enhanced gene transfection efficiency in cancer cells, but not in normal cells. It was
e-
also substantiated with in vivo experiments, in which a therapeutic DNA (i.e.,
Jo u
rn
al
Pr
pTRAIL) selectively induced apoptosis in subcutaneous A549 tumor-bearing mice.
Figure 8. Reaction oxygen species (ROS)-responsive charge-reversal polymer as non-viral gene delivery system. (a) Structure of B-PDEAEA and its ROS-induced charge reversal. (b) Formation of B-PDEAEA/DNA polyplexes and its ROS-triggered charge reversal to anionic polyacrylic acid. Reproduced with permission from Ref. [70]. Copyright 2016, John Wiley & Sons. 31
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4.2. Enzyme-triggered charge reversion from cationic to anionic In addition to killing cancer cells, chemotherapy was also found to induce adverse effect to fibroblasts by triggering a WNT16B pathway, which is accountable for tumor relapse and poor prognosis of chemotherapy [75]. Thus, it is desirable to design a drug delivery system which selectively exerts damaging effect to cancer cells, but not
oo
f
to fibroblasts. To this end, an esterase-responsive polymer (ERP), based on quaternary amines and possessing ester substitutes, was prepared as non-viral gene carriers [76].
pr
By taking advantage of the dramatic difference in esterase activity between cancer
e-
cells (i.e., HeLa) and fibroblasts (i.e., NIH3T3), the resulting ERP/pDNA polyplexes
Pr
promptly dissociated and released pDNA inside HeLa cells, owing to the
al
positive-to-negative charge reversal induced by the elevated esterase activity, but
rn
barely inside the fibroblasts. Consequently, the released pDNA encoding a cancer suicide gene (i.e., pTRAIL) was able to selectively kill cancer cells while induced
Jo u
minimal damaging effect to the fibroblasts, thus circumventing the WNT16B activation mechanism. The results show that such esterase-responsive gene vector was remarkably effective in inhibiting cancer cells in vitro and in vivo.
5.
Application of charge-reversal polyme rs (CRP) or components in the
fabrication of hybrid or liposomal nanocarriers for cancer therapy or diagnosis 5.1. Inorganic-organic hybrid micelles for siRNA delivery Besides its applications in fabricating polymeric micelles or polymer-drug conjugates, polymers possessing charge-reversal feature, or charge-reversal polymers 32
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(CRPs) are also useful in constructing organic- inorganic hybrid micelles. Given that calcium phosphate (CaP) has been extensively used in biomedical materials, i.e., drug/gene delivery systems, due to its advantages such as excelle nt biocompability, biodegradability and easy preparation [77-79], integration of biocompatible PEG-polypeptide and CaP into one hybrid delivery system, or one hybrid
f
naomedicine, should be highly beneficial for biomedical applications. Kataoka and
oo
coworkers took advantage of the anionic PEG-based CRP and siRNA to fabricate
pr
CRP/CaP/siRNA hybrid micelles [80-83], based on a notion that anionic species could
e-
inhibit the crystal growth of CaP NPs, resulting in the formation of hybrid
Pr
siRNA- loaded nanoparticles. First, together with anionic siRNA, a PEG-based block copolymer, with flanking amine groups on the side chain modified with aconitic
al
anhydride to confer negative charge, was used to complex with CaP to form
rn
CRP/siRNA/CaP hybrid nanocarriers. Such nanocarriers were characteristic of
Jo u
prolonged circulation time and excellent biocompatibility. Upon internalization into pancreatic cancer cell lines, i.e., PanC-1 and BxPC-3 cell lines, via endocytosis, the CRP within the hybrid nanocarriers underwent pH-dependent cleavage of amides and thus recovered the cationic amine moieties with endosomal membrane disrupting capability. Consequently, siRNA nanocarriers could escape from the acidic endo/lysosomal compartments and release siRNA into cytoplasm. Significant gene knockdown against endogenous VEGF was observed on pancreatic cancer cells, demonstrating great potential for siRNA delivery [80, 81]. In addition, the hybrid nanocarriers were used for the systemic siRNA delivery to spontaneous pancreatic 33
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tumors in EL1- Luc/TAg transgenic mice, in which increased accumulation of siRNA- loaded hybrid nanocarriers as well as remarked gene silencing again luciferase was observed.
These studies clearly demonstrated
the benefits of using
al
Pr
e-
pr
oo
f
charge-reversal siRNA/CaP hybrid nanocarriers in RNAi-based therapy [83].
rn
Figure 9. (A) Schematic illustration of hybrid micelles composed of PEG-CCP,
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siRNA, and CaP. (B) Chemical structure of PEG-PAsp(DET-Aco) (PEG-CCP). (C) Schematic illustration of the cellular delivery of siRNA by PEG-CCP/CaP hybrid micelles. Reproduced with permission from Ref. [81]. Copyright 2012, Elsevier.
5.2. Gold NPs coated with CRP for siRNA delivery Gold nanoparticles (AuNPs) have emerged as a promising delivery system for various payloads into cells, such as small drug molecules [84-88] or nucleic acids (i.e., pDNA and siRNA) [89-96]. Typically, nucleic acids were incorporated with AuNPs through a thiol linkage or electrostatic interaction with the cationic AuNPs. For example, a polycation, such as polyethyleneimine (PEI), was employed to coat onto 34
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the AuNP surface and subsequently bind with the anionic siRNA. Such siRNA- loaded AuNP exhibited strong endosomal escape capacity due to the proton sponge effect conferred by PEI. Unfortunately, low extent of siRNA was released into cytoplasm as a result of the high binding affinity between the AuNP and siRNA. Thus, increasing amount of siRNA is needed in order to achieve significant gene knockdown efficacy,
f
which is optimal for formulation.
oo
To address such a challenge, Liang et al. designed a type of ternary complexes
pr
composed of siRNA, a polycation, and a charge-reversal polymer [96]. Specifically, a
e-
charge-reversal siRNA nanocarrier was prepared using cationic PEI, charge-reversal
Pr
citraconic anhydride- modified poly(allylamine hydrochloride) (PAH-Cit), and mercaptoundecanoic acid-modified AuNPs (MUA-AuNPs) through a layer-by-layer technique.
Consequently,
PEI/PAH-Cit/PEI/AuNPs
were
obtained.
al
assembly
rn
Polyacrylamide gel electrophoresis (PAGE) was performed to determine the amount
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of siRNA released from the ternary AuNPs. The results showed that increasing amount of siRNA was released with increasing incubation time at pH 5.0 as a result of charge reversion of the Cit moiety in the siRNA/PEI/PAH-Cit/PEI/MUA-AuNP system, which is in contrast to control non-charge-reversal nanoparticles under identical conditions, i.e., siRNA/PEI/PSS/PEI/MUA-AuNPs. Furthermore, in vitro experiments, including gene transfection and siRNA knockdown studies, showed enhanced payload release and gene silencing efficacy due to the charge reversion of PAH-Cit. The advantage of such charge-reversal AuNPs as a siRNAtransfection agent was demonstrated by the observation that the charge-reversal AuNPs display higher 35
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siRNA transfection efficiency than the commercial transfection agents, such as Lipofectamine and PEI.
5.3. Upconversion NPs coated with CRP for diagnosis Photodynamic therapy (PDT) has emerged as a powerful strategy for cancer
f
treatment [97-99]. During the PDT treatment, ROS and singlet oxygen are generated
oo
upon light irradiation of a photosensitizer (PS) molecule at a selective wavelength,
pr
spanning from visible to NIR light. PDT is advantageous in that it can selectively kill
e-
cancer cells around the tumor lesion while minimizing damage to normal tissues.
Pr
More importantly, with the advances in PS synthesis, PDT can now be realized using an NIR light, in comparison to a conventional PDT treatment using visible or even
al
UV light. Thus, PDT using NIR-activation allows for much deeper tissue penetration
rn
and enables treatment of internal or large tumors [15, 72, 100].
Jo u
Upconversion nanoparticles (UCNPs) have demonstrated as promising materials for biomedical applications and in particular, have received tremendous attention as cancer nanomedicine [101-108]. In an upconversional system, absorption of two or more NIR photons leads to emission of a single photon with higher energy or a shorter wavelength. Such unique features can significantly address such issues as auto-fluorescence background, tissue penetration depth, and phototoxicity, which are commonly encountered in the traditional PDT treatment utilizing visible or UV light for excitation. Taking advantage of the merits of NIR-PDT and UCNPs, UCNPs have found tremendous potential in NIR-based PDT treatment of solid tumors [106, 109]. 36
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More recently, the development of smart dual- or multi- modal cancer nanomedicines, has opened up dramatic opportunities for UCNPs-based PDT. These new nanomedicines are advantageous over the conventional single- modal ones and should resolve the common issues of extended circulation and enhanced retentio n. Being able to
respond
to tumor-specific extracellular and/or
intracellular
f
microenvironment, smart UCNPs have shown significant enhancement in treatment
oo
efficacy for the in vivo PDT treatment of cancer in mice models.
pr
Liu and coworkers synthesized Mn2+-doped UCNPs emitting red light at ca. 660
e-
nm with an NIR excitation at 980 nm. The resulting UCNPs were subsequently used
Pr
to activate a photosensitizer, i.e., Chlorin e6 (Ce6), conjugated to cationic poly(allylamine hydrochloride) (PAH) to generate singlet oxygen for killing cancer
al
cells [106]. Ce6 molecules were loaded into the UCNPs through a layer-by-layer
rn
(LbL) approach, in which Ce6-PAH electrostatically complexed with anionic
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poly(acrylic acid) (PAA) decorated UCNPs. In order to confer negatively charged surface, DMMA modified PAH was used as a terminal coating for the NPs via LbL. The amide bonds underwent pH-dependent hydrolysis upon at the acid TME, which led to positively charged surface and dramatically enhanced cellular uptake of NPs, thus enhancing NIR- induced PDT efficacy in vitro. Due to Mn2+ doping, the UCNPs displayed optical and paramagnetic properties for in vivo dual- modal imaging after intratumoral injection. Overall, the Mn2+-doped UCNPs exhibited significantly enhanced PDT therapeutic efficacy in vivo, owing to the utilization of charge reversion polymer which conferred critical pH-dependent transformation for the 37
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UCNPs while at the tumor microenvironment.
5.4. Mesoporous silica nanoparticles (MSNs) coated with charge-reversal components to deliver anticancer agent Mesoporous silica nanoparticles (MSNs) have emerged as a promising candidate
f
for biomedical applications, in view of their excellent biocompatibility, high internal
oo
pore volume for drug loading, ease of functionalization, among others [110-112].
pr
Several surface modification strategies have been proposed to overcome challenges in
e-
the aspects of prolonged circulation, targetability, minimal non-specific adsorption of
Pr
proteins to the surface, among others. Although modification of MSN surface with zwitterionic species has demonstrated to be a useful approach toward stealth property,
al
such surface decoration may adversely affect the cellular internalization of the
rn
resulting MSNs. Therefore, a new strategy, which could address the above dilemma,
Jo u
is desirable. Chen et al. proposed to synthesize fluorescent MSNs which possessed switchable zwitterionic surface and were capped with polycaprolactone (PCL), which degrades at increased esterase activity inside cancer cells [113]. The resulting MSNs were loaded with an anticancer drug, Dox. Under physiological conditions, the MSNs exhibited excellent anti-biofouling property toward model plasma protein (i.e., bovine serum albumin) and esterase could not reach the PCL gate to trigger cargo release. While at acidic tumor milieu (pH<6.8), charge reversion took place, which diminished the anionic part of the zwitterionic surface and led to positively charged surface to facilitate cellular uptake by cancer cells. At the same time, due to enzymatic 38
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degradation of the PCL cap at the increasing presence of esterase in the MSN pore, intracellular release of Dox was triggered, as evidenced from the presence of free Dox in the nucleus and significantly enhanced cytotoxicity to cancer cells. The results clearly demonstrated that the synergetic effect induced by tumor acidity- induced charge reversal on the MSN surface and enzymatic degradation of PCL at pore gate
f
could effectively overcome the challenge of prolonged circulation time in the
oo
bloodstream and efficient cellular uptake and intracellular release of anticancer drug
pr
in tumor tissue, which is a common challenge in the application of MSNs as drug
Jo u
rn
al
Pr
e-
delivery systems.
Figure 10. Schematic representation of PCL/Dox- loaded fluorescent mesoporous silica nanocarriers with switchable zwitterionic surface and targeted drug delivery to cancer cells. Reproduced with permission from Ref. [113]. Copyright 2016, John Wiley & Sons.
5.5. Charge-reversal liposomes to deliver anticancer agent Liposomes, typically composed of phospholipids, are spherical vesicles of lipid bilayers and an aqueous core within the bilayers, which have demonstrated to be a 39
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safe and effective carrier system to incorporate hydrophobic and hydrophilic therapeutics [114-117]. In particular, liposomes have been widely used to deliver anticancer agents, i.e., small molecule drugs, nucleic acids and proteins, for cancer treatment. In order for better applicability in the systemic delivery of anticancer therapeutics, surface modification of liposomes with biocompatible PEG, or
f
PEGylation of liposomes, can lead to stealth liposomes, which display significantly
oo
improved blood circulation time and enhanced accumulation of the encapsulated
pr
anticancer agents into solid tumors through an enhanced permeability and retention
e-
(EPR) effect [118, 119]. Furthermore, liposomes can be modified with various ligands
Pr
to achieve specific targeting [120-122].
In view of the presence of biological challenges (i.e., improved blood circulation
al
and endosomal escape) during the course of systemic delivery, which could hinder the
liposomes
are
necessary.
To
that
end,
Mo
et
al.
prepared
a
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smart
rn
effective delivery of payload encapsulated within the liposomes, strategies toward
mitochondrial- targeted liposomal delivery system composed of a zwitterionic oligopeptide (HHG2C 18 -L), which exhibits multi-stage pH response to successively surpass a series of biological barriers in the systemic delivery of an anticancer drug, namely
temsirolimus
phosphatidylcholine,
[123].
The
cholesterol,
resulting and
liposomes a
synthetic
consist lipid
of
soy (i.e.,
1,5-dioctadecyl-L- glutamyl 2- histidyl- hexahydrobenzoic acid, HHG2C 18 ). The lipid carries a pH-cleavable group (i.e., hexahydrobenzoic amide) as a hydrophilic block, two stearyl alkane chains as a hydrophobic block, and two amino acids (i.e., glutamic 40
Journal Pre-proof
acid and histidine) to resemble the structure of natural phospholipids, which confers the resulting HHG2C18-L a multi- stage pH-responsive property, i.e., first to the mildly acidic extracellular tumor microenvironment and then to the acidic intracellular endo/lysosomal compartments. HHG2C18 -L displays negatively charged during blood circulation whereas it exhibits positively charged upon arrival at the mildly acidic
f
tumor microenvironment (i.e., around pH 6.5) due to the presence of zwitterionic
oo
oligopeptide. As a result of charge reversion or first-stage pH response, enhanced
pr
endocytosis took place. While at the acidic endo/lysosomal compartment, the histidine
e-
groups on the positively charged liposomes achieved proton sponge effect to facilitate
Pr
endosomal escape (i.e., second-stage pH response). Note that the pH-dependent hydrolysis, enabled by the pH-cleavable linker (i.e., hexahydrobenzoic amide) and
al
occurring in cytoplasm (i.e., third-stage pH response), was useful in maintaining the
rn
positive charge of HHG2C18 -L after its endo/lysosomal escape. It has been
Jo u
demonstrated that positive charge is critical to facilitate interactions with mitochondria to achieve mitochondria targeting. The results showed that, compared to non-pH-responsive controls, the introduction of charge reversion as well as multi-stage pH-response properties to HHG2C18-L significantly improved the therapeutic efficacy, as evidenced from the in vitro and in vivo experiments. In another study performed by Mo and coworkers [124], they demonstrated that the PEGylated HHG2C18-L also exhibited charge reversal and multi-stage pH responses to the mild acidic TME and the acidic endo/lysosomal compartments, successively. When encapsulated with anticancer agent (i.e., temsirolimus), the resulting 41
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drug- loaded liposomes displayed significantly enhanced tumor inhibition against xenograft renal cancer tumor models, thus validating the effectiveness of applying a
al
Pr
e-
pr
oo
f
charge-reversal strategy to liposomal delivery system.
Figure 11. Schematic representation of smart liposomes with multi-stage pH response
rn
to extracellular tumor microenvironment and intracellular compartments for drug
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delivery. Reproduced with permission from Ref. [123]. Copyright 2012, John Wiley & Sons.
6. Biosafety issues associated with charge-reversal nanocarriers Although the promising propensity realized by charge-reversal nanocarriers has triggered intense interest among the research community of cancer nanomedicine, the biosafety issues associated with charge reversal at biological or physiological environment are not fully understood yet and thus should deserve careful and systematic investigations. It is known that nanocarriers with positive surface charge are conducive to enhanced cellular uptake but they also induce undesired interaction 42
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with anionic serum components, leading to reticulo-endothelial system (RES) uptake in the liver and spleen (i.e., loss of injected dose), reduced circulation time and significant immune response. Such dilemma can be addressed by employing a charge-reversal strategy. Owing to the introduction of a charge-reversal moiety (i.e., negative-to-positive conversion) to the nanocarrier, the positive surface charge of
f
nanocarrier can be temporarily masked with a charge-reversal component to impart
oo
anionic or neutral feature during the blood circulation, which results in extended
pr
blood circulation time and minimal immune response. Up to date, most studies have
e-
only investigated the systemic toxicity or immune response after the systemic
Pr
administration of charge-reversal nanocarriers into mice. Given the fact that prior to negative-to-positive conversion, smart nanocarriers are neutral or anionic, which
al
usually exhibit excellent biocompatibility, owing to rational design of biocompatible
rn
nanocarriers. It, however, should be noted that upon the negative-to-positive
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conversion occurring at the desired site, i.e., tumor microenvironment, nanocarriers with cationic surface charge are recovered. Such a switch in surface charge can induce charge disturbance in the tumor microenvironment, which could, to some extent, trigger an adverse impact on the anionic cellular membrane [125, 126], the physiological polyanions (i.e., enzymes, cell receptors) [127], and possibly on the physiological functions of organs. Moreover, after charge reversal, the regenerated positively charged nanocarriers are expected to have a toxic effect, as typical cationic nanocarriers do. Unfortunately, to the best of our knowledge, there is no such study about the potential toxic effect or 43
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the immune response of charge-reversal nanocarriers after the negative-to-positive charge reversal takes place. In view of the critical importance associated with these biosafety issues, it is desirable that these biosafety issues should be thoroughly investigated in future studies.
Summary and outlook
f
7.
oo
Over the past decade or so, nanocarriers possessing charge-reversal property have
pr
received increasing attention in the research and development of cancer nanomedicine.
e-
The realization of charge reversal within a nanocarrier has demonstrated to play a
Pr
crucial role in concurrently achieving prolonged circulation time in the bloodstream and enhanced cellular internalization into cancer ce lls toward targeted release of
al
payloads to the intracellular compartments. Biological stimuli, including pH, redox,
rn
ROS or enzyme, can be readily harnessed to trigger charge reversion to confer
Jo u
nanocarriers favorable propensities toward smart cancer nanomedicine. Specifically, charge reversal taking place on the nanocarrier surface can effectively overcome certain benchmark biological barriers in the course of nanocarrier’s systemic transport and intracellular delivery, mainly including reticulo-endothelial system, cellular internalization, and endo/lysosomal membrane disruption. Such strategy is advantageous in that it is essential in enhancing therapeutic efficacy. Despite the aforementioned advantages, one should be aware of the disadvantages associated with charge-reversal nanocarriers. It is worth mentioning that for charge reversal triggered by pH, a precise control of pH-dependent cleavage or hydrolysis 44
Journal Pre-proof
within a narrow pH range is not easy, due to the complex nature of the biological or physiological environment. In addition, upon negative-to-positive charge reversal, the regeneration of the temporarily masked positive surface charge imparts the original core polymer or nanostructure cationic feature. Thus, drawbacks associated with cationic nanocarriers for systemic delivery may “relapse”. One would need to take
oo
development of practical cancer nanomedicine.
f
these factors into consideration when employing a charge-reversal strategy in the
pr
As far as diagnostic or theranostic nanocarriers are concerned, several critical
e-
features could be shared with the therapeutic counterparts to achieve maximum
Pr
efficacies. Thus, the design principle for therapeutic nanocarriers would be valid in such context. The charge-reversal strategy has also been successfully applied to the
al
fabrication of smart nanocarriers for cancer diagnostics or theranostics, which shows
rn
promising potential in improving efficacies. With the advancement in the precise
Jo u
synthesis of smart nanocarriers, it is necessary and viable to perform detailed investigations on how charge reversal, taking place in response to varying acidity of tumor extracellular or intracellular microenvironment, would be temporally influenced by difference in charge-reversal moiety. To this end, it is important to gain insights into how fast the chemical structure of pH- labile moieties modulates the charge reversal of resulting nanocarriers. Besides, owing to increasing understanding of cancer biology and with the advancement in nano-biotechnology, it can be envisioned that charge-reversal nanocarriers would continue to be an important arena in the research field of cancer nanomedicine and thus deserves thorough explorations 45
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toward their full potential as precision nanomedicine for cancer diagnosis and treatment. In view of the extensive merits observed in the laboratory research for charge-reversal nanocarriers versus their traditional counterparts, it would be reasonable for one to anticipate improved benefits for their potential applications in clinic settings. In this sense, one may be cautiously optimistic about the clinical
oo
f
applicability and benefits of charge-reversal cancer nanomedicine.
pr
Declarations of interest: none.
e-
Acknowledgements
Pr
This work was supported in part by the National Natural Science Foundation of China (grant numbers: 51173035, 31670922 and 81771636), the Major Project of
al
Wenzhou Science & Technology Bureau (grant numbers ZS2017012, Y20160069),
References
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rn
and a grant from the 2nd Affiliated Hospital of Wenzhou Medical University.
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Journal Pre-proof Table and Figures
Table 1 Stimulus
Site of action for charge reversal
Ref.
Protein
pH
IC
25-28
pDNA Drug
pH pH
IC IC
29-30 35, 53
Drug Drug
pH pH
IC and TME TME
51 52
Drug Drug
pH pH
Drug
Nanogels
Drug
Hybrid micelles
siRNA
Gold NPs Upconversion NPs
siRNA PS
MSNs
Drug
Liposomes Positive-toNegative
Polymer micelles
IC IC，TME
36 50
Enzyme
TME
62
pH
TME
37
pH
IC
80-83
pH pH
TME TME
96 106
pH
TME
113
Drug
pH
TME
124, 125
Drug
ROS
IC
70
Drug
Enzyme
IC
76
oo
Positive
Polymer-drug conjugates
al
Negative-to-
pr
Polymer micelles
f
Payload
e-
Nanocarrier composition
Pr
Type of charge reversal
Jo u
rn
IC: Intracellular compartments TME: Extracellular tumor microenvironment PS: Photosensitizer MSNs: Mesoporous silica nanoparticles
59
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
al
Figure 1
60
Journal Pre-proof
(a)
pr
oo
f
(b)
Jo u
rn
al
Pr
e-
Figure 2
61
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
al
Pr
Figure 3
62
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
Figure 4
63
al
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
Figure 5
64
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
al
Figure 6
65
Figure 7
Jo u
rn
al
Pr
e-
pr
oo
f
Journal Pre-proof
66
Jo u
rn
al
Pr
e-
pr
oo
f
Journal Pre-proof
Figure 8
67
al
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
Figure 9
68
Jo u
rn
al
Pr
e-
pr
Figure 10
oo
f
Journal Pre-proof
69
Jo u
rn
al
Pr
e-
pr
oo
f
Journal Pre-proof
Figure 11
70
Jo u
rn
al
Pr
e-
pr
oo
f
Journal Pre-proof
71
al
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
Figure 1
72
Journal Pre-proof
(a)
pr
oo
f
(b)
Jo u
rn
al
Pr
e-
Figure 2
73
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
al
Pr
Figure 3
74
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
Figure 4
75
al
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
Figure 5
76
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
al
Figure 6
77
Figure 7
Jo u
rn
al
Pr
e-
pr
oo
f
Journal Pre-proof
78
Jo u
rn
al
Pr
e-
pr
oo
f
Journal Pre-proof
Figure 8
79
al
Pr
e-
pr
oo
f
Journal Pre-proof
Jo u
rn
Figure 9
80
Jo u
rn
al
Pr
e-
pr
Figure 10
oo
f
Journal Pre-proof
81
Jo u
rn
al
Pr
e-
pr
oo
f
Journal Pre-proof
Figure 11
82
Journal Pre-proof
Payload
Stimulus
Site of action for charge reversal
Ref.
Protein
pH
IC
25-28
pDNA
pH
IC
29-30
Drug
pH
IC
35, 53
Drug
pH
IC and TME
51
Drug Drug
pH pH
TME IC
52 36
conjugates
Drug Drug
pH Enzyme
IC，TME
50 62
Nanogels Hybrid micelles
Drug siRNA
pH pH
TME IC
37 80-83
Gold NPs
siRNA
pH
TME
96
Upconversion NPs
PS
pH
TME
106
MSNs
Drug
pH
TME
113
Liposomes
Drug Drug
pH ROS
TME IC
124, 125 70
Enzyme
IC
76
Positive-toNegative
Polymer-drug
Polymer micelles
Drug
Pr
Negative-toPositive
IC: Intracellular compartments
TME
oo
Polymer micelles
pr
Nanocarrier composition
e-
Type of charge reversal
f
Table 1
al
TME: Extracellular tumor microenvironment PS: Photosensitizer
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Highlights
rn
MSNs: Mesoporous silica nanoparticles
Different strategies for charge-reversal cancer nanomedicine are discussed.
Stealth and extended circulation properties can be achieved in systemic applications. Charge reversal at tumor extracellular environment leads to enhanced cellular uptake.
Charge reversal is conducive to enhanced intracellular delivery of therapeutics.
83