- Email: [email protected]
Construction and evaluation of PAMAM–DOX conjugates with superior tumor recognition and intracellular acid-triggered drug release properties
Accepted Manuscript Title: Construction and Evaluation of PAMAM-DOX Conjugates with Superior Tumor Recognition and Intracellular Acid-Triggered Drug Release Properties Author: Lifang Cheng Qing Hu Liang Cheng Wen Hu Ming Xu Yaqin Zhu Lu Zhang Dawei Chen PII: DOI: Reference:
S0927-7765(15)00209-X http://dx.doi.org/doi:10.1016/j.colsurfb.2015.04.003 COLSUB 7006
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
13-10-2014 17-3-2015 1-4-2015
Please cite this article as: L. Cheng, Q. Hu, L. Cheng, W. Hu, M. Xu, Y. Zhu, L. Zhang, D. Chen, Construction and Evaluation of PAMAM-DOX Conjugates with Superior Tumor Recognition and Intracellular Acid-Triggered Drug Release Properties, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.04.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
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1. PAMAM-DOX conjugates (FPP-hyd-DOX) with different FA ratios were
3
constructed and identified.
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2. In-vitro evaluations exhibited the conjugates release DOX in an obvious
5
pH-triggered manner.
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3. FPP-hyd-DOX was internalized by KB cells via clathrin-mediated endocytosis.
7
4. Subcellular localization study revealed drug release of FPP-hyd-DOX was
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triggered in lysosomes.
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5. FPP-hyd-DOX conjugations would be a promising drug delivery carrier for
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targeted cancer therapy.
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Construction and Evaluation of PAMAM-DOX Conjugates with
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Superior Tumor Recognition and Intracellular Acid-Triggered Drug
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Release Properties
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Lifang Chenga,c, Qing Hua,c, Liang Chenga, Wen Hua, Ming Xua, Yaqin Zhua,
16
Lu Zhanga, Dawei Chen a,b,*
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a
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b
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c
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*Corresponding Author: Dawei Chen, Ph.D., Prof.
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Mailing address: College of Pharmaceutical Science, Soochow University, 199 Ren’ai
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Road, Suzhou 215123, P.R. China
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Telephone number: +86 512-6588-4729; Fax: +86 24-2398-6250
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E-mail: [email protected]
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Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China. School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China.
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Both authors contributed equally to this work.
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ABSTRACT An ideal drug delivery system for cancer therapy should be equipped with
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extended circulation, improved tumor targeting and controlled drug release, as well as
29
low toxicity from the carrier. In this study, a multifunctional drug delivery system
30
based on the PEGylated poly(amidoamine) (PAMAM) dendrimer was designed, and
31
folate-PEGylation was applied to modify the dendrimer in order to enhance tumor
32
selectivity. A series of acid-labile PAMAM-DOX conjugates (FPP-hyd-DOX) with
33
different FA ligand ratios were successfully constructed. 1H NMR, FTIR, DLS and
34
TEM were used to describe the physicochemical characterization of PAMAM-DOX
35
conjugates. Both in vitro drug release assay and subcellular localization, the
36
conjugates exhibited an obvious pH-triggered drug release. The FPP-hyd-DOX 16/1
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displayed much lower IC50 value than that of non-targeted PP-hyd-DOX. Through
38
fluoresence microscopy and flow cytometry investigations, the cellular uptake of
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FPP-hyd-DOX 16/1 was obviously enhanced, compared with that of PP-hyd-DOX.
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The cellular uptake mechanism and subcellular localization study revealed that the conjugates were internalized by KB cells via FA receptor and clathrin co-mediated endocytosis, delivered to acidic lysosomes and triggered the release of DOX into nuclei to exert its cytotoxicity. These obtained results showed that FPP-hyd-DOX
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conjugations would be a promising drug delivery carrier for targeted cancer therapy.
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KEY WORDS PAMAM; pH-sensitive; active targeting; doxorubicin; tumor
46
microenvironment
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1 Introduction 3
Page 3 of 34
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Cancer is regarded as one of the most dangerous diseases for human health. The
49
traditional therapy for cancer including chemotherapy, radiotherapy, biological
50
immune methods, etc, of which chemotherapy is the main method in clinic study [1-2].
51
However,
52
pharmacokinetics profiles and/or are distributed nonspecifically in the body, leading
53
to systemic toxicity and unsatisfactory cure rate. An ideal drug delivery system should
54
have the responsibility to overcome these problems to increase the patients’ survival
55
time and life quality.
the
conventional
chemotherapeutic
agents
have
poor
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of
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In cancer therapy, the tumor microenvironment is one of many areas which are
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studied to design new therapies, especially attempts at novel nanotechnology-based
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therapies [2]. Compared with the normal tissue, the tumor microenvironment has
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unique characteristics, which could be used to design special drug delivery system
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(DDS) [3]. These distinguishing features include the formation of a vascular network
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with poorly constructed and highly leaky architecture, the lack of a functioning
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lymphatic supply, the decreased extracellular pH (pH 5.5-7.0), intracellular acidic organelles (pH 4.5-6.0 in lysosomes ) and the increased expression of various antigens, receptors and enzymes by the tumor cells [4, 5]. Based on these, various targeting mechanisms, including passive targeting via EPR effects, receptor-mediated active
66
targeting and environment-sensitive drug controlled release, have been widely applied
67
in the design of smart DDS [6].
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Dendrimers are highly branched macromolecules possessing well-defined
69
nanoscale architecture, multivalency, and structural versatility, have been regarded as 4
Page 4 of 34
a promising class of nanobiomaterials for drug delivery [7, 8]. PAMAM dendrimers,
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the first complete dendrimer family to be synthesized, characterized and
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commercialized, have been utilized as delivery carriers for gene, chemotherapy drug
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and imaging agents [9-15]. However, the charge-related toxicity and high risk of
74
clearance by reticuloendothelial system (RES), limited their biomedical applications
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[16]. To overcome these drawbacks, an ideal DDS based on PAMAM should be
76
equipped with extended circulation, improved tumor targeting and controlled drug
77
release in targeted cancer cells.
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Biocompatible poly(ethylene glycol) (PEG) is often used to overcome the
79
toxicity problems, minimize aggregation of nanoparticles and improve the solubility
80
of drug delivery system. PEGylated dendrimer is characteristic of long circulation
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time which could avoid the rapid clearance by the reticuloendothelial system (RES)
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and increase tumor effective accumulation through the enhanced permeability and
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retention (EPR) effect [17-19]. Besides, PEG chains could be conveniently modified
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with ligands to target specific tissues and tumors. For example, folate receptor (FR) expression is usually amplified in a variety of human cancers though concomitantly restricted in most normal tissues [10, 20]. Introduction of folate (FA) molecules to the end of PEG chains can induce a targeting property to the carrier for its efficient endocytosis into FR-bearing tumor cells [21-25].
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Chemotherapeutic drugs can be associated with PAMAM via physical
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encapsulation of drugs within the structure, or via covalent conjugation to the surface.
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Physical encapsulation has some disadvantages, such as the burst leakage of drug in 5
Page 5 of 34
blood circulation and poorly controlled release at the desired tissue, often leading to
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decrease of in vivo therapeutic effect. In contrast, covalent attachments via
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pH-sensitive or enzyme-degradable linkages are able to facilitate drug release in a
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predicted and controlled fashion [4, 26]. The acidic tumor microclimate is the most
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common characteristic of solid tumor [5], so incorporating acid-sensitive linkage
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between the drug and the dendrimer has been an attractive and increasingly employed
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strategy for triggered release of the dendrimer-bound drug at the tumor site. The
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acid-triggered release could occur either in extracellular matrix, the slightly acidic
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condition in tumor tissue, or within tumor cells, in acidic lysosomes/endosomes after
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cellular uptake [27]. Acid-sensitive linkages including hydrazone [28], cis-aconityl
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[29] and esters spacers [3] have been exploited for pH-trigger drug release, of which,
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hydrazone linkage is an ideal chemical bond for conjugation of chemotherapeutic
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drugs with dendrimer. This linkage can ensure effective release of the polymer-bound
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drug in the acidic cellular compartments after internalization by tumor cells, while
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keeping the conjugates stable and intact in the bloodstream [29]. The purpose of this study was to construct a smart DDS based on G4 PAMAM
with reduced cytotoxicity of PAMAM, increased tumor targeting ability, and intracellular microenvironment-triggered drug release properties. A series of
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acid-labile PAMAM-DOX conjugates with different number of FA ligands,
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PP-hyd-DOX, FPP-hyd-DOX 4/1, FPP-hyd-DOX 8/1 and FPP-hyd-DOX 16/1, were
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constructed and evaluated. Doxorubicin (DOX), a widely used antitumor drug, was
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coupled to the surface of PAMAM dendrimers through a pH-sensitive hydrazone bond 6
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(hyd) for controlled drug release, PEG chains were introduced for long circulation,
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and FA was conjugated to PEG for receptor-mediated active targeting (Scheme 1).
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The 1H NMR, FTIR, DLS and TEM analyses were used to characterize the conjugates.
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In vitro drug release test was performed in different pH conditions to evaluate release
118
mechanism of the conjugates. The targeting effect was revealed by MTT experiments,
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fluorescence microscopy, flow cytometry and confocal laser scanning microscopy.
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The uptake mechanism of the PAMAM-DOX conjugates by KB cells was also
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illustrated in the presence of endocytosis inhibitors.
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2 Materials and methods
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2.1 Materials
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G4 PAMAM dendrimer was purchased from Dendritech, Inc. Michigan (Midland,
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MI, USA). Methoxy PEG succinimidyl carboxymethyl ester (mPEG-NHS，Mw =
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5000) and amine PEG amine (NH2-PEG-NH2, Mw = 5147) were obtained from
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JenKem
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Technology
Co.
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Ltd.
(Beijing,
China).
Folic
acid
(FA),
p-nitrophenylchloroformate, 85% hydrazine hydrate aqueous solution, triethylamine (TEA), N-hydroxysuccinimide (NHS), N,N-dicyclohexyl carbon diimine (DCC), Disuccinimidyl suberate (DSS) was obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Doxorubicin Hydrochloride (DOX・HCl) was purchased from
132
Beijing
HuaFeng
United
Technology
Co.
Ltd.
(Beijing,
China).
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Hoechst
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33342 was obtained from Shanghai Beyotime Biotechnology Co. Ltd. (Suzhou,
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China). 7
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2.2 Synthesis of conjugates
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2.2.1 Synthesis of FA-PEG-NHS The folate-poly(ethylene glycol)-hydroxysuccinimide (FA-PEG-NHS) was
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prepared using the previously reported method with minor modification [30]. Briefly,
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FA (0.048 mM), DCC (0.06 mmol) and NHS (0.06 mM) were dissolved in DMSO (5
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ml), in the presence of TEA (0.31 mM) and the solution was stirred for 6 h at room
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temperature in the dark to produce the active ester of FA, which was constantly
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dropwise added to a solution of NH2-PEG-NH2 (0.04 mM) in DMSO. After overnight
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stirring in the dark, the precipitated side-product dicyclohexylurea (DCU) and
145
triethylamine were removed by filter and evaporation under reduced pressure,
146
respectively. The product was dialyzed against Na2CO3-NaHCO3 (0.1 M) for 2 days
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to remove free FA, desalted over deionized water, and finally lyophilized to yield the
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FA-PEG-NH2 as a pale yellow solid. The yellow solid was dissolved in DMSO (5.0
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ml) containing triethylamine (4.0 µl) as a base, and a ten-fold excess of DSS was
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added. After overnight stirring, the product was dialyzed against deionized water, and then purified through a Sephadex G-50 column (1.0 × 80 cm, GE Healthcare, Sweden).
2.2.2 Synthesis of PEG-PAMAM
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The mPEG-NHS was added to a solution of PAMAM in 4 ml phosphate buffer
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(0.1 M, pH 8.2), with a molar ratio between PEG and PAMAM of 16:1. The reaction
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mixtures were incubated at room temperature for 24 h with stirring. Thin layer
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chromatography (TLC) was used to monitor the reaction process. The solution was 8
Page 8 of 34
dialyzed against deionized water for 2 days to remove unreacted PEG. The products
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were retrieved by freeze drying, and obtained white solids. The synthesized
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PEG-PAMAM conjugates were characterized by 1H NMR in D2O. PEGylation degree
161
was estimated using the proton integration method.
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2.2.3 Synthesis of PEG-PAMAM-hyd-DOX (PP-hyd-DOX) Conjugates
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The PP-hyd-DOX conjugates were synthesized using the method by Yoo with
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modification [31]. PEG-PAMAM was firstly activated by p-nitrophenylchloroformate
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in 5 ml of methylene chloride solution containing 4.8 µl of pyridine at 4 °C for 2 h.
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Later, the reaction was carried out for another 24 h at room temperature under
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nitrogen atmosphere. The activated PEG-PAMAM was recovered by precipitation in
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ice-cold diethyl ether and dried under reduced pressure. 85% hydrazine hydrate
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aqueous solution (27 mM) was added to a solution of activated PEG-PAMAM in
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CH3OH. The mixture was refluxed for 24 h at 70 °C, then concentrated by rotary
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evaporation, dialyzed against deionized water, and freeze-dried. Further reactions
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were carried out by mixing the hydrazine conjugated PEG-PAMAM with DOX dissolved in DMF in the presence of triethylamine (4 µl). The reaction was allowed to proceed for 24 h at 60 °C under nitrogen atmosphere. The product was purified by gel filtration on a Sephadex LH-20 column (3.5 × 60 cm, GE Healthcare, Sweden)
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equilibrated with CH3OH to remove unreacted DOX and dried under reduced pressure.
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The conjugated number of DOX per PAMAM dendrimer was determined by
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UV-visible spectroscopy at 491 nm (UV-2600, Japan).
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2.2.4 Synthesis of FA-PEG-PAMAM-hyd-DOX (FPP-hyd-DOX) Conjugates 9
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Generation 4 PAMAM dendrimer was firstly modified with mPEG-NHS to
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produce PEG-PAMAM conjugates. Then DOX was coupled to the PEG-PAMAM by
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hydrazone linkage to form PP-hyd-DOX. Finally, certain molar ratio of FA-PEG-NHS
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was added to PP-hyd-DOX through the amidation reaction.
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PAMAM-DOX conjugates with different FA ligand numbers (PP-hyd-DOX,
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FPP-hyd-DOX 4/1, FPP-hyd-DOX 8/1 and FPP-hyd-DOX 16/1) were prepared by
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adjusting the feed ratio of FA-PEG-NHS, mPEG-NHS and PP-hyd-DOX.
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FA-PEG-NHS and PEG-PAMAM-hyd-DOX with the molar ratio of 4:1 were
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dissolved in phosphate buffer solution (0.1 M, pH 8.2). After 12 h of reaction at room
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temperature, mPEG-NHS (mPEG-NHS: PEG-PAMAM-hyd-DOX = 12:1, molar ratio)
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were added and then the reaction carried out for another 12 h to produce
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FPP-hyd-DOX 4/1. The synthetic method of other PAMAM-DOX conjugates were
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similar to FPP-hyd-DOX 4/1 described above, but the molar ratio of FA-PEG-NHS,
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mPEG-NHS and PEG-PAMAM-hyd-DOX was different. FPP-hyd-DOX 8/1
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(FA-PEG-NHS:
PEG-PAMAM-hyd-DOX
PEG-PAMAM-hyd-DOX
=
8:1,
(FA-PEG-NHS:PEG-PAMAM-hyd-DOX
molar =
= ratio),
16:1,
molar
8:1,
mPEG-NHS:
FPP-hyd-DOX ratio),
16/1
PP-hyd-DOX
(,mPEG-NHS:PEG-PAMAM-hyd-DOX = 16:1, molar ratio, without FA-PEG-NHS). 2.3 Dynamic Light Scattering (DLS) and Zeta Potential Measurement
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The synthesized products were dissolved in 0.1 M PBS (pH 7.4, 0.5 mg/ml) and
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filtered through a 0.22 µm cellulose acetate membrane. Then, the particle size and
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Zeta potential were determined by DLS using PSS•Nicomp 380 ZLS (Santa Barbara 10
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California, USA). All measurements were carried out for 3 times.
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2.4 Transmission Electron Micrograph (TEM) Morphology TEM measurement was performed on a TecnaiG220 electronic microscopy to
205
observe the size and morphology of the sample. A drop of PP-hyd-DOX or
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FPP-hyd-DOX 16/1 solution (0.5 mg/ml) was placed on the carbon-coated Formvar
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copper grid for 2 min, and then the grid was tapped with filter paper to remove the
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excess aqueous solution and air-dried.
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2.5 In Vitro Release of DOX from PAMAM-DOX Conjugates
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The release studies were performed at 37 °C in acetate buffer (0.1 M, pH 4.5 and
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5.5) and PBS (0.1 M, pH 7.4) solutions, respectively. PAMAM-DOX conjugates (10
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mg) were separately dispersed in 2 ml of medium and placed in a dialysis bag
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(MWCO 3500). The dialysis bag was then immersed into 25 ml of the same buffer
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solution and kept in a constant temperature (37 °C) and stirring speed (120 rpm). 1 ml
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of release medium was taken out at various time and replaced by 1 ml of fresh buffer
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solution. The released DOX was calculated with a standard curve draw by the HPLC method (Promosil ODS C18 column (4.6 × 250 mm, 5 μm particle size), 0.01 M NH4H2PO4 (adjusted to pH 3.0 using acetic acid): acetonitrile = 35: 65 (V/V), 1.0 ml/min, 30 °C, 490 nm). 2.6 Cell Culture
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KB cells were obtained from Shanghai Institute of Cell Biology. They were
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cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
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of fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin sulfate. 11
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All the cells were cultured at 37 °C with 5% CO2 under fully humidified conditions.
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2.7 In Vitro Cytotoxicity Studies To test the targeting selectivity, KB cells were seeded in 96-well plates at a
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density of 5×103 cells/well and incubated for 24 h. Then the conjugates (DOX,
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PP-hyd-DOX, FPP-hyd-DOX 4/1, FPP-hyd-DOX 8/1, and FPP-hyd-DOX 16/1) were
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mixed with DMEM medium to a 5 µM DOX concentration and added immediately to
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cells. After 48 h incubation, 10 µl of MTT (5 mg/ml) was added to medium and then
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the cells were incubated for another 4 h. Afterwards 100 µL of DMSO was added to
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dissolve the precipitates and the resulting solution was measured by a microplate
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reader at 492 nm (Multiskan MK3, China).
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The cytotoxicity of the conjugates against KB cells was assessed by MTT assay.
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KB cells were seeded at a density of 5×103 cells/well in 96-well plates and incubated
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for 24 h. Then PAMAM, FA-PEG-PAMAM, free DOX, PP-hyd-DOX and
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FPP-hyd-DOX 16/1were added, with the final PAMAM and DOX concentration
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d
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ranging from 0 to 100 µM, respectively. After 48 h, the cells were treated as described above. The IC50 values were expressed as concentration (μM) of PAMAM dendrimer-equiv or DOX-equiv. 2.8 Cellular Uptake of PAMAM-DOX conjugates
242
KB cells were seeded at a density of 1×104 cells/well in 24-well plates and
243
incubated for 48 h. Serum-free culture media with PP-hyd-DOX and FPP-hyd-DOX
244
(5 µM DOX-equiv) were added and the cells were further incubated for 1 h and 2 h.
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After removing the solution, Hoechst 33342 (10 µg/ml) was added and incubated for 12
Page 12 of 34
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another 15 min. Then the cells were washed three times with cold PBS then visualized
247
under fluorescent microscope (IX51, Olympus, Japan) [32]. Quantitative analysis of the cellular uptake was carried out using flow cytometry
249
method (FCM). KB cells were seeded at a density of 2×104 cells/well in 6-well plates
250
and incubated for 24 h. After adding free DOX, PP-hyd-DOX and FPP-hyd-DOX (10
251
µM DOX-equiv) to the cells, cells were incubated for another 1 h and 2 h. Then, the
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cells were washed three times with cold PBS and harvested, which were subsequently
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resuspended in PBS and analyzed using flow cytometer (Beckman Coulter, America).
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2.9 Route of Cellular Uptake
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To study the effect of various endocytosis inhibitors on the uptake of
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PAMAM-DOX conjugates, KB cells were firstly pre-incubated with the following
257
inhibitors individually: chlorpromazine (10 μg/ml), colchicines (40 μg/ml), filipin (4
258
μg/ml), polylysine (PLL, 800 μg/ml) and FA (500 μg/ml) [26, 30, 31]. The MTT assay
259
was employed to determine the cytotoxicity of the endocytosis inhibitors. For the
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inhibition study, KB cells were pre-incubated with various inhibitors for 30 min at 37 °C, respectively. Then, FPP-hyd-DOX and PP-hyd-DOX (10 μM DOX-equiv.) were added and incubated for another 1 h. Cells were washed and harvested for flow cytometry analysis as described above. 2.10 Subcellular Localization of the PAMAM-DOX Conjugates
265
KB cells were grown on 22-mm glass coverslip in 6-well plates at a density of
266
2×104 cells/well and incubated for 24 h. DOX (0.1 µM), PP-hyd-DOX,
267
FPP-hyd-DOX (10 µM DOX-equiv) in serum-free culture media were added and the 13
Page 13 of 34
cells were further incubated for 2 h. After incubation, the drug-containing solutions
269
were removed, and Lysotracker Green DND-26 (80 nM, 1 h) and Hoechst 33342 (10
270
µg/ml, 15 min) were used to visualize endosome/lysosome and nuclei, respectively.
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Afterwards, cells were washed 3 times with cold PBS before fixing with 4%
272
paraformaldehyde for 20 min at room temperature. Images of treated cells were
273
obtained by a confocal laser scanning microscope (TCS-SP2, Leica, Germany).
274
Chloroquine (8 μg/ml), a lysosomal pH-enhancing agent, was added to the culture
275
dishes for 1 h at 37 °C before the addition of FPP-hyd-DOX 16/1 [18].
276
2.11 Statistics Analysis
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One-way analysis of variance (ANOVA) was used to determine significance
278
among groups following the Bonferroni’s post-test. Data were presented as mean ±
279
standard deviation (SD). P value of 0.05 or less was considered to be statistically
280
significant.
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3 Results and discussion
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3.1 Synthesis and Characterization of PAMAM-DOX Conjugates FA was conjugated to NH2-PEG-NH2 and then activated by DSS to produce
FA-PEG-NHS. FTIR (Fig. S1) and 1H NMR were used to characterize the obtained compounds. Fig. 1A was the 1H NMR spectrum of FA-PEG-NHS. The peaks at 8.62,
286
7.67, 6.80 ppm and 3.3~3.8 ppm were attributed to FA segments and the protons of
287
CH2CH2O repeat units of PEG, respectively. The mPEG-NHS chains were grafted on
288
G4 PAMAM by the amidation reaction. TLC, FTIR (Fig. S2) and 1H NMR were
289
employed to characterize PEG-PAMAM. The 1H NMR spectrum of PEG-PAMAM 14
Page 14 of 34
conjugate was shown in Fig. 1B. The PEGylation was confirmed by the signal
291
appeared at 3.4~3.6 in 1H NMR spectra of the conjugates, corresponding to the
292
protons of CH2CH2O repeat units. Using the proton integration method, it could be
293
calculated that the conjugated number of PEG chain on PEG-PAMAM was 12. As
294
shown in Fig. 1C, the signals (1.71~2.73 ppm) were attributable to PAMAM, and the
295
signals at 8.75, 7.65, 6.75 and 6.5~8.5 ppm were assigned to the protons FA units and
296
DOX, respectively. The number of FA per PAMAM was determined to be 10.1 on
297
average by comparing the integrals of signal at 8.75 ppm and at 1.71 ppm. The 1H
298
NMR spectrums of FPP-hyd-DOX 4/1 and FPP-hyd-DOX 8/1 (Fig. S3) were similar
299
to that of FPP-hyd-DOX 16/1, and the average FA number was 3.3 and 5.8,
300
respectively. The substitution level of DOX on the NH2 of PAMAM was determined
301
to be 6.11 (wt. %) by UV-vis, which corresponds to 10 DOX molecules per PAMAM
302
on average.
304 305 306 307
cr
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FPP-hyd-DOX conjugates with different number of FA ligands were synthesized
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by controlling the feed ratios of the starting materials. The conjugated numbers of FA molecule were estimated to be 3.3, 5.8 and 10.1 per PAMAM molecular corresponding to the feed molar ratios of FA-PEG-NHS and PP-hyd-DOX were 4:1, 8:1 and 16:1, respectively (Table 1). DLS was performed to characterize the size and
308
Zeta potential of PAMAM and PAMAM-DOX conjugates. All the PAMAM-DOX
309
conjugates showed increased particle size and decreased Zeta potential compared with
310
unmodified PAMAM dendrimer (Table 1). The conjugates, with the size ranging from
311
~20 to ~30 nm (data by volume), could avoid the fast elimination by RES and 15
Page 15 of 34
facilitate the permeation and accumulation in tumor tissues. TEM images displayed
313
spherical shape for both PP-hyd-DOX (Fig. 2A) and FPP-hyd-DOX 16/1 (Fig. 2B).
314
Compared with PAMAM dendrimer (+18.90 ± 0.11 mv), the Zeta potential of
315
conjugates modified with PEG decreased to about +5 mv as previously presented,
316
which was considered to be helpful to cellular uptake as confirmed by Zhu et al [28].
317
3.2 In Vitro Release of DOX from PAMAM-DOX Conjugates
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To investigate the acid-sensitivity of PAMAM-DOX conjugates, the release
319
profiles were conducted at pH 4.5, pH 5.5 and pH 7.4. The DOX release rate was
320
dramatically improved as the pH value decreased. As shown in Fig. 3, at pH 7.4, the
321
hydrazone linker kept stable, and all the conjugates released less than 8% of the total
322
DOX over 48 h, whereas progressive decrease of pH from 7.4 to 4.5 resulted in
323
obvious DOX release increase. The 48 h accumulative drug release percentages of
324
PP-hyd-DOX, FPP-hyd-DOX 4/1, 8/1 and 16/1 at pH 5.5 increased to 28.72±0.52%,
325
28.97±0.92%, 27.24±1.05% and 27.97±0.81%, respectively, while the corresponding
327 328 329
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releases at pH 4.5 reached up to 43.05±0.69%, 42.46±0.95%, 42.03±1.07% and 41.46±0.52% of total DOX. From the results, all the PAMAM-DOX conjugates, PP-hyd-DOX, FPP-hyd-DOX 4/1, 8/1 and 16/1, displayed acidic triggered drug release profiles, which directly proved the intracellular microenvironment (as in
330
lysosomes pH4.5~6.5) cleavable characteristic of the hydrazone linkage between
331
PAMAM and DOX as presented by Hu [35]. Besides, modification of FA did not
332
influence the in vitro release behavior of PAMAM-DOX conjugates, and all the
333
conjugates with different numbers of FA displayed similar release profiles. The 16
Page 16 of 34
maximal drug release from the PAMAM-DOX conjugates previously described by Li
335
was 32% at pH 4.5 within 48 h [36], while in our study the release was over 40%
336
under the same condition. But similar to Li, no more apparent DOX release was
337
observed over 48 h, which was presumed that hydrated layer formed by PEG chains
338
on the periphery of the conjugates blocked the drug cut from hydrazone bound to
339
penetrate. The findings agreed with those reported by Shen [6] who had verified that
340
PEG layer had certain effect on the drug release. Due to the molecular structural
341
characteristics of PAMAM and solubility of DOX, we presumed that part of the
342
released DOX from FPP-hyd-DOX was potentially re-encapsulated into the
343
hydrophobic core of PAMAM dendrimer.
344
3.3 In Vitro Cytotoxicity Studies
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The cytotoxicity of free DOX, blank carrier and PAMAM-DOX conjugates
346
against KB cells were studied using the MTT assay. As shown in Fig. 4A, all the
347
FPP-hyd-DOX exhibited higher cytotoxicity than PP-hyd-DOX, and the cell viability
349 350 351
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decreased with the number of FA ligands increasing, indicating that FA promoted the uptake of conjugates into the FR-overexpressed cancer cells and thus increased the intracellular drug accumulation. FPP-hyd-DOX 16/1 was determined to be the optimal formulation with the highest uptake and cytotoxicity, thus was selected to do
352
the following experiments. The IC50 value of FPP-hyd-DOX 16/1 (2.77 µM) was
353
2.2-fold lower than that of non-targeted conjugate PP-hyd-DOX (6.18 µM) (Fig. 4 C).
354
The viability of KB cells after 48 h incubation with blank carriers was shown in Fig.
355
4B. PAMAM dendrimers showed significant cytotoxicity against KB cells with an 17
Page 17 of 34
IC50 value of 2.33 μM. Introduction of FA-PEG-NHS reduced the cytotoxicity of
357
PAMAM, and more than half of the cells were still alive even at the highest
358
concentration (100 μM).
359
3.4 Cellular Uptake of PAMAM-DOX conjugates
ip t
356
To evaluate the effect of FA on targeting capacity, fluorescence microscopy was
361
used to study the cellular uptake characteristics. The results were shown in Fig. 5A-D,
362
as the incubation time increased, both the fluorescence intensity of KB cells treated
363
with FPP-hyd-DOX 16/1 and PP-hyd-DOX increased. At either 1 h or 2 h, the
364
fluorescence intensity of KB cells treated with FPP-hyd-DOX 16/1 was obviously
365
higher than that treated with PP-hyd-DOX.
M
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The quantitative analysis of cellular uptake was further investigated using FCM.
367
Flow cytometry histograms of cell-associated DOX fluorescence against KB cells
368
were shown in Fig. 5E-F. With the same incubation time and DOX concentration,
369
FPP-hyd-DOX 16/1 showed higher fluorescence intensity than free DOX and
371 372 373
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PP-hyd-DOX. After 2 h incubation, the FPP-hyd-DOX 16/1 showed a 1.4-fold increase in cellular uptake than that of PP-hyd-DOX. Free DOX was found to be similar in uptake with PP-hyd-DOX. These results suggested that receptor-mediated endocytosis played important role in the cellular uptake and FA ligands attaching on
374
the surface of conjugates significantly promoted the uptake [37].
375
3.5 Route of Cellular Uptake
376
Various uptake inhibitors, incuding Chlorpromazine, colchicines, filipin, PLL
377
and FA [37-40], were used to study the uptake route of PAMAM-DOX conjugates by 18
Page 18 of 34
KB cells. Many of previous reports have investigated the cellular internalization
379
mechanism of PAMAM dendrimers. The obtained results seemed to be dependent on
380
the cell type, the surface modification and particle size [15, 40, 41]. As shown in Fig.
381
6A, each inhibitor displayed no obvious cytotoxicity against KB cells. Fig. 6B
382
presented the cell uptake of PAMAM-DOX conjugates in the presence of various
383
inhibitors. Chlorpromazine decreased the cellular uptake of FPP-hyd-DOX 16/1 and
384
PP-hyd-DOX to 63.8% and 66.6% of the control, respectively, indicating that
385
clathrin-mediated endocytosis was blocked by disturbing the formation of
386
clathrin-coated pits [26, 30, 36]. Incubation with PLL reduced the cellular uptake of
387
FPP-hyd-DOX 16/1 and PP-hyd-DOX to 76.8% and 73.2% of the control,
388
respectively, displaying the importance of surface positive charge on the
389
internalization of PAMAM-DOX conjugates [39, 42, 43]. The effect of
390
caveolae-mediated endocytosis on the internalization of the conjugates was evaluated
391
using filipin, and no significant uptake difference was observed compared with each
393 394 395
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control. The minimal effect of colchicines on conjugates uptake showed that macro-pinocytosis was presumably involved to a lesser extent. Free FA decreased cellular uptake of FPP-hyd-DOX 16/1 to 82.4%, but had no effect on PP-hyd-DOX uptake, suggesting that FA-FR recognition was involved in the cellular uptake of
396
FPP-hyd-DOX 16/1 by FR overexpressing cells. Endocytosis inhibition experiments
397
demonstrated that PAMAM-DOX conjugates were internalized by KB cells mainly
398
through FR and clathria co-mediated endocytosis, while the contribution of
399
caveolae-mediated endocytosis and macro-pinocytosis was minimal. 19
Page 19 of 34
400
3.6 Subcellular Localization of the PAMAM-DOX Conjugates The subcellular localization of the conjugates in KB cells was evaluated by
402
confocal laser scanning microscopy (CLSM). As shown in Fig. 6C, free DOX, the red
403
fluorescence, was found to localize in the nuclei of KB cells, showing a
404
co-localization with the blue fluorescence of Hoechst 33342 (Fig. 6C a-d). As to
405
FPP-hyd-DOX 16/1 and PP-hyd-DOX, DOX related red fluorescence could be
406
observed both in the lysosomes and nuclei, displaying co-localization with
407
LysoTracker green (a specific marker for lysosome) and Hoechst 33342 (Fig. 6C e-h
408
and Fig. 6C i-l). The internalization of the FPP-hyd-DOX 16/1 was much higher than
409
that of PP-hyd-DOX. Chloroquine was applied to enhance the pH value of the
410
lysosome compartment of KB cells. As a result, no red fluorescence could be
411
observed in the nuclei, indicating that the acidic condition was essential for DOX
412
release from FPP-hyd-DOX 16/1 (Fig. 6C m-p). Confocal images showed that the
413
conjugates were delivered to lysosome where the slightly acidic conditions triggered
415 416 417
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the release of DOX, and then the released DOX was transported into the nuclei to exert anti-tumor effects [35]. 4 Conclusions
In this study, acid-labile PAMAM-DOX conjugates with different number of FA
418
ligands were successfully synthesized. Both in vitro drug release assay and CLSM
419
images exhibited that DOX release from the PAMAM-DOX conjugates depended
420
strongly on the pH values. As in lysosomes (pH4.5~6.5), DOX could be fast released
421
from the conjugates. Compared with non-targeted PP-hyd-DOX, FPP-hyd-DOX 16/1 20
Page 20 of 34
had obviously greater cytotoxicity and cellular uptake against KB cells, due to the
423
FR-mediated active targeting. Endocytosis mechanism studies further revealed that
424
the PAMAM-DOX conjugates were internalized by KB cells mainly through
425
clathrin-mediated endocytosis, and transported to lysosomes, where DOX was
426
released from the conjugates and subsequently entered into the nucleus. Overall, the
427
present study demonstrates that the FA-modified PAMAM-DOX conjugates possess
428
excellent targeting ability and intracellular pH-responsive drug release property.
429
Thereby, it may be a promising drug delivery carrier for targeted cancer therapy.
430
Acknowledgements
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The work was supported by National Natural Science Foundation of China
432
(81173004 and 81302719) and PAPD (A Project Funded by the Priority Academic
433
Program Developments of Jiangsu Higher Education Institutions, China).
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Page 21 of 34
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M
509
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506
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510
25
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510 511 512
Number of
Feed ratio
Number of
ip t
Table 1 Characteristics of PAMAM-DOX conjugates Particle size
Zeta potential
FA
FA:PAMAM
DOX
1
( H NMR)
(by UV-vis)
PAMAM
-
-
-
PP-hyd-DOX
-
-
FPP-hyd-DOX 4/1
4:1
3.3
FPP-hyd-DOX 8/1
8:1
5.8
FPP-hyd-DOX 16/1
16:1
10.1
an
(mv) (n=3)
(by volume)
us
(by mol)
(nm) (n=3)
cr
Conjugates
5.1 ± 0.1
18.90 ± 0.11
20.1 ± 0.3
4.53 ± 0.03
25.3 ± 0.2
4.79 ± 0.13
23.4 ± 0.3
4.67 ± 0.07
30.3 ± 0.9
4.88 ± 0.05
d te
514 515
Ac ce p
513
M
10.1
26
Page 26 of 34
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Figure 1
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Figure 2
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ed
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Figure 3
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Figure 4
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ed
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Figure 5
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Figure 6
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Graphical Abstract (for review)
Page 33 of 34
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Scheme 1
Page 34 of 34
S0927-7765(15)00209-X http://dx.doi.org/doi:10.1016/j.colsurfb.2015.04.003 COLSUB 7006
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
13-10-2014 17-3-2015 1-4-2015
Please cite this article as: L. Cheng, Q. Hu, L. Cheng, W. Hu, M. Xu, Y. Zhu, L. Zhang, D. Chen, Construction and Evaluation of PAMAM-DOX Conjugates with Superior Tumor Recognition and Intracellular Acid-Triggered Drug Release Properties, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.04.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
2
1. PAMAM-DOX conjugates (FPP-hyd-DOX) with different FA ratios were
3
constructed and identified.
4
2. In-vitro evaluations exhibited the conjugates release DOX in an obvious
5
pH-triggered manner.
6
3. FPP-hyd-DOX was internalized by KB cells via clathrin-mediated endocytosis.
7
4. Subcellular localization study revealed drug release of FPP-hyd-DOX was
8
triggered in lysosomes.
9
5. FPP-hyd-DOX conjugations would be a promising drug delivery carrier for
cr
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targeted cancer therapy.
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1
11
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1
Page 1 of 34
Construction and Evaluation of PAMAM-DOX Conjugates with
13
Superior Tumor Recognition and Intracellular Acid-Triggered Drug
14
Release Properties
15
Lifang Chenga,c, Qing Hua,c, Liang Chenga, Wen Hua, Ming Xua, Yaqin Zhua,
16
Lu Zhanga, Dawei Chen a,b,*
cr
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12
17 18
a
19
b
20
c
21
*Corresponding Author: Dawei Chen, Ph.D., Prof.
22
Mailing address: College of Pharmaceutical Science, Soochow University, 199 Ren’ai
23
Road, Suzhou 215123, P.R. China
24
Telephone number: +86 512-6588-4729; Fax: +86 24-2398-6250
25
E-mail: [email protected]
us
Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China. School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China.
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Both authors contributed equally to this work.
2
Page 2 of 34
26
ABSTRACT An ideal drug delivery system for cancer therapy should be equipped with
28
extended circulation, improved tumor targeting and controlled drug release, as well as
29
low toxicity from the carrier. In this study, a multifunctional drug delivery system
30
based on the PEGylated poly(amidoamine) (PAMAM) dendrimer was designed, and
31
folate-PEGylation was applied to modify the dendrimer in order to enhance tumor
32
selectivity. A series of acid-labile PAMAM-DOX conjugates (FPP-hyd-DOX) with
33
different FA ligand ratios were successfully constructed. 1H NMR, FTIR, DLS and
34
TEM were used to describe the physicochemical characterization of PAMAM-DOX
35
conjugates. Both in vitro drug release assay and subcellular localization, the
36
conjugates exhibited an obvious pH-triggered drug release. The FPP-hyd-DOX 16/1
37
displayed much lower IC50 value than that of non-targeted PP-hyd-DOX. Through
38
fluoresence microscopy and flow cytometry investigations, the cellular uptake of
39
FPP-hyd-DOX 16/1 was obviously enhanced, compared with that of PP-hyd-DOX.
41 42 43
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40
ip t
27
The cellular uptake mechanism and subcellular localization study revealed that the conjugates were internalized by KB cells via FA receptor and clathrin co-mediated endocytosis, delivered to acidic lysosomes and triggered the release of DOX into nuclei to exert its cytotoxicity. These obtained results showed that FPP-hyd-DOX
44
conjugations would be a promising drug delivery carrier for targeted cancer therapy.
45
KEY WORDS PAMAM; pH-sensitive; active targeting; doxorubicin; tumor
46
microenvironment
47
1 Introduction 3
Page 3 of 34
48
Cancer is regarded as one of the most dangerous diseases for human health. The
49
traditional therapy for cancer including chemotherapy, radiotherapy, biological
50
immune methods, etc, of which chemotherapy is the main method in clinic study [1-2].
51
However,
52
pharmacokinetics profiles and/or are distributed nonspecifically in the body, leading
53
to systemic toxicity and unsatisfactory cure rate. An ideal drug delivery system should
54
have the responsibility to overcome these problems to increase the patients’ survival
55
time and life quality.
the
conventional
chemotherapeutic
agents
have
poor
ip t
of
an
us
cr
most
In cancer therapy, the tumor microenvironment is one of many areas which are
57
studied to design new therapies, especially attempts at novel nanotechnology-based
58
therapies [2]. Compared with the normal tissue, the tumor microenvironment has
59
unique characteristics, which could be used to design special drug delivery system
60
(DDS) [3]. These distinguishing features include the formation of a vascular network
61
with poorly constructed and highly leaky architecture, the lack of a functioning
63 64 65
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62
M
56
lymphatic supply, the decreased extracellular pH (pH 5.5-7.0), intracellular acidic organelles (pH 4.5-6.0 in lysosomes ) and the increased expression of various antigens, receptors and enzymes by the tumor cells [4, 5]. Based on these, various targeting mechanisms, including passive targeting via EPR effects, receptor-mediated active
66
targeting and environment-sensitive drug controlled release, have been widely applied
67
in the design of smart DDS [6].
68
Dendrimers are highly branched macromolecules possessing well-defined
69
nanoscale architecture, multivalency, and structural versatility, have been regarded as 4
Page 4 of 34
a promising class of nanobiomaterials for drug delivery [7, 8]. PAMAM dendrimers,
71
the first complete dendrimer family to be synthesized, characterized and
72
commercialized, have been utilized as delivery carriers for gene, chemotherapy drug
73
and imaging agents [9-15]. However, the charge-related toxicity and high risk of
74
clearance by reticuloendothelial system (RES), limited their biomedical applications
75
[16]. To overcome these drawbacks, an ideal DDS based on PAMAM should be
76
equipped with extended circulation, improved tumor targeting and controlled drug
77
release in targeted cancer cells.
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Biocompatible poly(ethylene glycol) (PEG) is often used to overcome the
79
toxicity problems, minimize aggregation of nanoparticles and improve the solubility
80
of drug delivery system. PEGylated dendrimer is characteristic of long circulation
81
time which could avoid the rapid clearance by the reticuloendothelial system (RES)
82
and increase tumor effective accumulation through the enhanced permeability and
83
retention (EPR) effect [17-19]. Besides, PEG chains could be conveniently modified
85 86 87 88
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84
M
78
with ligands to target specific tissues and tumors. For example, folate receptor (FR) expression is usually amplified in a variety of human cancers though concomitantly restricted in most normal tissues [10, 20]. Introduction of folate (FA) molecules to the end of PEG chains can induce a targeting property to the carrier for its efficient endocytosis into FR-bearing tumor cells [21-25].
89
Chemotherapeutic drugs can be associated with PAMAM via physical
90
encapsulation of drugs within the structure, or via covalent conjugation to the surface.
91
Physical encapsulation has some disadvantages, such as the burst leakage of drug in 5
Page 5 of 34
blood circulation and poorly controlled release at the desired tissue, often leading to
93
decrease of in vivo therapeutic effect. In contrast, covalent attachments via
94
pH-sensitive or enzyme-degradable linkages are able to facilitate drug release in a
95
predicted and controlled fashion [4, 26]. The acidic tumor microclimate is the most
96
common characteristic of solid tumor [5], so incorporating acid-sensitive linkage
97
between the drug and the dendrimer has been an attractive and increasingly employed
98
strategy for triggered release of the dendrimer-bound drug at the tumor site. The
99
acid-triggered release could occur either in extracellular matrix, the slightly acidic
100
condition in tumor tissue, or within tumor cells, in acidic lysosomes/endosomes after
101
cellular uptake [27]. Acid-sensitive linkages including hydrazone [28], cis-aconityl
102
[29] and esters spacers [3] have been exploited for pH-trigger drug release, of which,
103
hydrazone linkage is an ideal chemical bond for conjugation of chemotherapeutic
104
drugs with dendrimer. This linkage can ensure effective release of the polymer-bound
105
drug in the acidic cellular compartments after internalization by tumor cells, while
107 108 109
cr
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d
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Ac ce p
106
ip t
92
keeping the conjugates stable and intact in the bloodstream [29]. The purpose of this study was to construct a smart DDS based on G4 PAMAM
with reduced cytotoxicity of PAMAM, increased tumor targeting ability, and intracellular microenvironment-triggered drug release properties. A series of
110
acid-labile PAMAM-DOX conjugates with different number of FA ligands,
111
PP-hyd-DOX, FPP-hyd-DOX 4/1, FPP-hyd-DOX 8/1 and FPP-hyd-DOX 16/1, were
112
constructed and evaluated. Doxorubicin (DOX), a widely used antitumor drug, was
113
coupled to the surface of PAMAM dendrimers through a pH-sensitive hydrazone bond 6
Page 6 of 34
(hyd) for controlled drug release, PEG chains were introduced for long circulation,
115
and FA was conjugated to PEG for receptor-mediated active targeting (Scheme 1).
116
The 1H NMR, FTIR, DLS and TEM analyses were used to characterize the conjugates.
117
In vitro drug release test was performed in different pH conditions to evaluate release
118
mechanism of the conjugates. The targeting effect was revealed by MTT experiments,
119
fluorescence microscopy, flow cytometry and confocal laser scanning microscopy.
120
The uptake mechanism of the PAMAM-DOX conjugates by KB cells was also
121
illustrated in the presence of endocytosis inhibitors.
122
2 Materials and methods
123
2.1 Materials
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114
G4 PAMAM dendrimer was purchased from Dendritech, Inc. Michigan (Midland,
125
MI, USA). Methoxy PEG succinimidyl carboxymethyl ester (mPEG-NHS，Mw =
126
5000) and amine PEG amine (NH2-PEG-NH2, Mw = 5147) were obtained from
127
JenKem
129 130 131
te
Technology
Co.
Ac ce p
128
d
124
Ltd.
(Beijing,
China).
Folic
acid
(FA),
p-nitrophenylchloroformate, 85% hydrazine hydrate aqueous solution, triethylamine (TEA), N-hydroxysuccinimide (NHS), N,N-dicyclohexyl carbon diimine (DCC), Disuccinimidyl suberate (DSS) was obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Doxorubicin Hydrochloride (DOX・HCl) was purchased from
132
Beijing
HuaFeng
United
Technology
Co.
Ltd.
(Beijing,
China).
133
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Hoechst
134
33342 was obtained from Shanghai Beyotime Biotechnology Co. Ltd. (Suzhou,
135
China). 7
Page 7 of 34
136
2.2 Synthesis of conjugates
137
2.2.1 Synthesis of FA-PEG-NHS The folate-poly(ethylene glycol)-hydroxysuccinimide (FA-PEG-NHS) was
139
prepared using the previously reported method with minor modification [30]. Briefly,
140
FA (0.048 mM), DCC (0.06 mmol) and NHS (0.06 mM) were dissolved in DMSO (5
141
ml), in the presence of TEA (0.31 mM) and the solution was stirred for 6 h at room
142
temperature in the dark to produce the active ester of FA, which was constantly
143
dropwise added to a solution of NH2-PEG-NH2 (0.04 mM) in DMSO. After overnight
144
stirring in the dark, the precipitated side-product dicyclohexylurea (DCU) and
145
triethylamine were removed by filter and evaporation under reduced pressure,
146
respectively. The product was dialyzed against Na2CO3-NaHCO3 (0.1 M) for 2 days
147
to remove free FA, desalted over deionized water, and finally lyophilized to yield the
148
FA-PEG-NH2 as a pale yellow solid. The yellow solid was dissolved in DMSO (5.0
149
ml) containing triethylamine (4.0 µl) as a base, and a ten-fold excess of DSS was
151 152 153
cr
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d
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Ac ce p
150
ip t
138
added. After overnight stirring, the product was dialyzed against deionized water, and then purified through a Sephadex G-50 column (1.0 × 80 cm, GE Healthcare, Sweden).
2.2.2 Synthesis of PEG-PAMAM
154
The mPEG-NHS was added to a solution of PAMAM in 4 ml phosphate buffer
155
(0.1 M, pH 8.2), with a molar ratio between PEG and PAMAM of 16:1. The reaction
156
mixtures were incubated at room temperature for 24 h with stirring. Thin layer
157
chromatography (TLC) was used to monitor the reaction process. The solution was 8
Page 8 of 34
dialyzed against deionized water for 2 days to remove unreacted PEG. The products
159
were retrieved by freeze drying, and obtained white solids. The synthesized
160
PEG-PAMAM conjugates were characterized by 1H NMR in D2O. PEGylation degree
161
was estimated using the proton integration method.
162
2.2.3 Synthesis of PEG-PAMAM-hyd-DOX (PP-hyd-DOX) Conjugates
cr
ip t
158
The PP-hyd-DOX conjugates were synthesized using the method by Yoo with
164
modification [31]. PEG-PAMAM was firstly activated by p-nitrophenylchloroformate
165
in 5 ml of methylene chloride solution containing 4.8 µl of pyridine at 4 °C for 2 h.
166
Later, the reaction was carried out for another 24 h at room temperature under
167
nitrogen atmosphere. The activated PEG-PAMAM was recovered by precipitation in
168
ice-cold diethyl ether and dried under reduced pressure. 85% hydrazine hydrate
169
aqueous solution (27 mM) was added to a solution of activated PEG-PAMAM in
170
CH3OH. The mixture was refluxed for 24 h at 70 °C, then concentrated by rotary
171
evaporation, dialyzed against deionized water, and freeze-dried. Further reactions
173 174 175
an
M
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Ac ce p
172
us
163
were carried out by mixing the hydrazine conjugated PEG-PAMAM with DOX dissolved in DMF in the presence of triethylamine (4 µl). The reaction was allowed to proceed for 24 h at 60 °C under nitrogen atmosphere. The product was purified by gel filtration on a Sephadex LH-20 column (3.5 × 60 cm, GE Healthcare, Sweden)
176
equilibrated with CH3OH to remove unreacted DOX and dried under reduced pressure.
177
The conjugated number of DOX per PAMAM dendrimer was determined by
178
UV-visible spectroscopy at 491 nm (UV-2600, Japan).
179
2.2.4 Synthesis of FA-PEG-PAMAM-hyd-DOX (FPP-hyd-DOX) Conjugates 9
Page 9 of 34
Generation 4 PAMAM dendrimer was firstly modified with mPEG-NHS to
181
produce PEG-PAMAM conjugates. Then DOX was coupled to the PEG-PAMAM by
182
hydrazone linkage to form PP-hyd-DOX. Finally, certain molar ratio of FA-PEG-NHS
183
was added to PP-hyd-DOX through the amidation reaction.
ip t
180
PAMAM-DOX conjugates with different FA ligand numbers (PP-hyd-DOX,
185
FPP-hyd-DOX 4/1, FPP-hyd-DOX 8/1 and FPP-hyd-DOX 16/1) were prepared by
186
adjusting the feed ratio of FA-PEG-NHS, mPEG-NHS and PP-hyd-DOX.
187
FA-PEG-NHS and PEG-PAMAM-hyd-DOX with the molar ratio of 4:1 were
188
dissolved in phosphate buffer solution (0.1 M, pH 8.2). After 12 h of reaction at room
189
temperature, mPEG-NHS (mPEG-NHS: PEG-PAMAM-hyd-DOX = 12:1, molar ratio)
190
were added and then the reaction carried out for another 12 h to produce
191
FPP-hyd-DOX 4/1. The synthetic method of other PAMAM-DOX conjugates were
192
similar to FPP-hyd-DOX 4/1 described above, but the molar ratio of FA-PEG-NHS,
193
mPEG-NHS and PEG-PAMAM-hyd-DOX was different. FPP-hyd-DOX 8/1
195 196 197 198
us
an
M
d
te
Ac ce p
194
cr
184
(FA-PEG-NHS:
PEG-PAMAM-hyd-DOX
PEG-PAMAM-hyd-DOX
=
8:1,
(FA-PEG-NHS:PEG-PAMAM-hyd-DOX
molar =
= ratio),
16:1,
molar
8:1,
mPEG-NHS:
FPP-hyd-DOX ratio),
16/1
PP-hyd-DOX
(,mPEG-NHS:PEG-PAMAM-hyd-DOX = 16:1, molar ratio, without FA-PEG-NHS). 2.3 Dynamic Light Scattering (DLS) and Zeta Potential Measurement
199
The synthesized products were dissolved in 0.1 M PBS (pH 7.4, 0.5 mg/ml) and
200
filtered through a 0.22 µm cellulose acetate membrane. Then, the particle size and
201
Zeta potential were determined by DLS using PSS•Nicomp 380 ZLS (Santa Barbara 10
Page 10 of 34
202
California, USA). All measurements were carried out for 3 times.
203
2.4 Transmission Electron Micrograph (TEM) Morphology TEM measurement was performed on a TecnaiG220 electronic microscopy to
205
observe the size and morphology of the sample. A drop of PP-hyd-DOX or
206
FPP-hyd-DOX 16/1 solution (0.5 mg/ml) was placed on the carbon-coated Formvar
207
copper grid for 2 min, and then the grid was tapped with filter paper to remove the
208
excess aqueous solution and air-dried.
209
2.5 In Vitro Release of DOX from PAMAM-DOX Conjugates
an
us
cr
ip t
204
The release studies were performed at 37 °C in acetate buffer (0.1 M, pH 4.5 and
211
5.5) and PBS (0.1 M, pH 7.4) solutions, respectively. PAMAM-DOX conjugates (10
212
mg) were separately dispersed in 2 ml of medium and placed in a dialysis bag
213
(MWCO 3500). The dialysis bag was then immersed into 25 ml of the same buffer
214
solution and kept in a constant temperature (37 °C) and stirring speed (120 rpm). 1 ml
215
of release medium was taken out at various time and replaced by 1 ml of fresh buffer
217 218 219 220
d
te
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216
M
210
solution. The released DOX was calculated with a standard curve draw by the HPLC method (Promosil ODS C18 column (4.6 × 250 mm, 5 μm particle size), 0.01 M NH4H2PO4 (adjusted to pH 3.0 using acetic acid): acetonitrile = 35: 65 (V/V), 1.0 ml/min, 30 °C, 490 nm). 2.6 Cell Culture
221
KB cells were obtained from Shanghai Institute of Cell Biology. They were
222
cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
223
of fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin sulfate. 11
Page 11 of 34
224
All the cells were cultured at 37 °C with 5% CO2 under fully humidified conditions.
225
2.7 In Vitro Cytotoxicity Studies To test the targeting selectivity, KB cells were seeded in 96-well plates at a
227
density of 5×103 cells/well and incubated for 24 h. Then the conjugates (DOX,
228
PP-hyd-DOX, FPP-hyd-DOX 4/1, FPP-hyd-DOX 8/1, and FPP-hyd-DOX 16/1) were
229
mixed with DMEM medium to a 5 µM DOX concentration and added immediately to
230
cells. After 48 h incubation, 10 µl of MTT (5 mg/ml) was added to medium and then
231
the cells were incubated for another 4 h. Afterwards 100 µL of DMSO was added to
232
dissolve the precipitates and the resulting solution was measured by a microplate
233
reader at 492 nm (Multiskan MK3, China).
M
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The cytotoxicity of the conjugates against KB cells was assessed by MTT assay.
235
KB cells were seeded at a density of 5×103 cells/well in 96-well plates and incubated
236
for 24 h. Then PAMAM, FA-PEG-PAMAM, free DOX, PP-hyd-DOX and
237
FPP-hyd-DOX 16/1were added, with the final PAMAM and DOX concentration
239 240 241
te
Ac ce p
238
d
234
ranging from 0 to 100 µM, respectively. After 48 h, the cells were treated as described above. The IC50 values were expressed as concentration (μM) of PAMAM dendrimer-equiv or DOX-equiv. 2.8 Cellular Uptake of PAMAM-DOX conjugates
242
KB cells were seeded at a density of 1×104 cells/well in 24-well plates and
243
incubated for 48 h. Serum-free culture media with PP-hyd-DOX and FPP-hyd-DOX
244
(5 µM DOX-equiv) were added and the cells were further incubated for 1 h and 2 h.
245
After removing the solution, Hoechst 33342 (10 µg/ml) was added and incubated for 12
Page 12 of 34
246
another 15 min. Then the cells were washed three times with cold PBS then visualized
247
under fluorescent microscope (IX51, Olympus, Japan) [32]. Quantitative analysis of the cellular uptake was carried out using flow cytometry
249
method (FCM). KB cells were seeded at a density of 2×104 cells/well in 6-well plates
250
and incubated for 24 h. After adding free DOX, PP-hyd-DOX and FPP-hyd-DOX (10
251
µM DOX-equiv) to the cells, cells were incubated for another 1 h and 2 h. Then, the
252
cells were washed three times with cold PBS and harvested, which were subsequently
253
resuspended in PBS and analyzed using flow cytometer (Beckman Coulter, America).
254
2.9 Route of Cellular Uptake
an
us
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248
To study the effect of various endocytosis inhibitors on the uptake of
256
PAMAM-DOX conjugates, KB cells were firstly pre-incubated with the following
257
inhibitors individually: chlorpromazine (10 μg/ml), colchicines (40 μg/ml), filipin (4
258
μg/ml), polylysine (PLL, 800 μg/ml) and FA (500 μg/ml) [26, 30, 31]. The MTT assay
259
was employed to determine the cytotoxicity of the endocytosis inhibitors. For the
261 262 263 264
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260
M
255
inhibition study, KB cells were pre-incubated with various inhibitors for 30 min at 37 °C, respectively. Then, FPP-hyd-DOX and PP-hyd-DOX (10 μM DOX-equiv.) were added and incubated for another 1 h. Cells were washed and harvested for flow cytometry analysis as described above. 2.10 Subcellular Localization of the PAMAM-DOX Conjugates
265
KB cells were grown on 22-mm glass coverslip in 6-well plates at a density of
266
2×104 cells/well and incubated for 24 h. DOX (0.1 µM), PP-hyd-DOX,
267
FPP-hyd-DOX (10 µM DOX-equiv) in serum-free culture media were added and the 13
Page 13 of 34
cells were further incubated for 2 h. After incubation, the drug-containing solutions
269
were removed, and Lysotracker Green DND-26 (80 nM, 1 h) and Hoechst 33342 (10
270
µg/ml, 15 min) were used to visualize endosome/lysosome and nuclei, respectively.
271
Afterwards, cells were washed 3 times with cold PBS before fixing with 4%
272
paraformaldehyde for 20 min at room temperature. Images of treated cells were
273
obtained by a confocal laser scanning microscope (TCS-SP2, Leica, Germany).
274
Chloroquine (8 μg/ml), a lysosomal pH-enhancing agent, was added to the culture
275
dishes for 1 h at 37 °C before the addition of FPP-hyd-DOX 16/1 [18].
276
2.11 Statistics Analysis
an
us
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268
One-way analysis of variance (ANOVA) was used to determine significance
278
among groups following the Bonferroni’s post-test. Data were presented as mean ±
279
standard deviation (SD). P value of 0.05 or less was considered to be statistically
280
significant.
281
3 Results and discussion
283 284 285
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M
277
3.1 Synthesis and Characterization of PAMAM-DOX Conjugates FA was conjugated to NH2-PEG-NH2 and then activated by DSS to produce
FA-PEG-NHS. FTIR (Fig. S1) and 1H NMR were used to characterize the obtained compounds. Fig. 1A was the 1H NMR spectrum of FA-PEG-NHS. The peaks at 8.62,
286
7.67, 6.80 ppm and 3.3~3.8 ppm were attributed to FA segments and the protons of
287
CH2CH2O repeat units of PEG, respectively. The mPEG-NHS chains were grafted on
288
G4 PAMAM by the amidation reaction. TLC, FTIR (Fig. S2) and 1H NMR were
289
employed to characterize PEG-PAMAM. The 1H NMR spectrum of PEG-PAMAM 14
Page 14 of 34
conjugate was shown in Fig. 1B. The PEGylation was confirmed by the signal
291
appeared at 3.4~3.6 in 1H NMR spectra of the conjugates, corresponding to the
292
protons of CH2CH2O repeat units. Using the proton integration method, it could be
293
calculated that the conjugated number of PEG chain on PEG-PAMAM was 12. As
294
shown in Fig. 1C, the signals (1.71~2.73 ppm) were attributable to PAMAM, and the
295
signals at 8.75, 7.65, 6.75 and 6.5~8.5 ppm were assigned to the protons FA units and
296
DOX, respectively. The number of FA per PAMAM was determined to be 10.1 on
297
average by comparing the integrals of signal at 8.75 ppm and at 1.71 ppm. The 1H
298
NMR spectrums of FPP-hyd-DOX 4/1 and FPP-hyd-DOX 8/1 (Fig. S3) were similar
299
to that of FPP-hyd-DOX 16/1, and the average FA number was 3.3 and 5.8,
300
respectively. The substitution level of DOX on the NH2 of PAMAM was determined
301
to be 6.11 (wt. %) by UV-vis, which corresponds to 10 DOX molecules per PAMAM
302
on average.
304 305 306 307
cr
us
an
M
d
te
FPP-hyd-DOX conjugates with different number of FA ligands were synthesized
Ac ce p
303
ip t
290
by controlling the feed ratios of the starting materials. The conjugated numbers of FA molecule were estimated to be 3.3, 5.8 and 10.1 per PAMAM molecular corresponding to the feed molar ratios of FA-PEG-NHS and PP-hyd-DOX were 4:1, 8:1 and 16:1, respectively (Table 1). DLS was performed to characterize the size and
308
Zeta potential of PAMAM and PAMAM-DOX conjugates. All the PAMAM-DOX
309
conjugates showed increased particle size and decreased Zeta potential compared with
310
unmodified PAMAM dendrimer (Table 1). The conjugates, with the size ranging from
311
~20 to ~30 nm (data by volume), could avoid the fast elimination by RES and 15
Page 15 of 34
facilitate the permeation and accumulation in tumor tissues. TEM images displayed
313
spherical shape for both PP-hyd-DOX (Fig. 2A) and FPP-hyd-DOX 16/1 (Fig. 2B).
314
Compared with PAMAM dendrimer (+18.90 ± 0.11 mv), the Zeta potential of
315
conjugates modified with PEG decreased to about +5 mv as previously presented,
316
which was considered to be helpful to cellular uptake as confirmed by Zhu et al [28].
317
3.2 In Vitro Release of DOX from PAMAM-DOX Conjugates
us
cr
ip t
312
To investigate the acid-sensitivity of PAMAM-DOX conjugates, the release
319
profiles were conducted at pH 4.5, pH 5.5 and pH 7.4. The DOX release rate was
320
dramatically improved as the pH value decreased. As shown in Fig. 3, at pH 7.4, the
321
hydrazone linker kept stable, and all the conjugates released less than 8% of the total
322
DOX over 48 h, whereas progressive decrease of pH from 7.4 to 4.5 resulted in
323
obvious DOX release increase. The 48 h accumulative drug release percentages of
324
PP-hyd-DOX, FPP-hyd-DOX 4/1, 8/1 and 16/1 at pH 5.5 increased to 28.72±0.52%,
325
28.97±0.92%, 27.24±1.05% and 27.97±0.81%, respectively, while the corresponding
327 328 329
M
d
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Ac ce p
326
an
318
releases at pH 4.5 reached up to 43.05±0.69%, 42.46±0.95%, 42.03±1.07% and 41.46±0.52% of total DOX. From the results, all the PAMAM-DOX conjugates, PP-hyd-DOX, FPP-hyd-DOX 4/1, 8/1 and 16/1, displayed acidic triggered drug release profiles, which directly proved the intracellular microenvironment (as in
330
lysosomes pH4.5~6.5) cleavable characteristic of the hydrazone linkage between
331
PAMAM and DOX as presented by Hu [35]. Besides, modification of FA did not
332
influence the in vitro release behavior of PAMAM-DOX conjugates, and all the
333
conjugates with different numbers of FA displayed similar release profiles. The 16
Page 16 of 34
maximal drug release from the PAMAM-DOX conjugates previously described by Li
335
was 32% at pH 4.5 within 48 h [36], while in our study the release was over 40%
336
under the same condition. But similar to Li, no more apparent DOX release was
337
observed over 48 h, which was presumed that hydrated layer formed by PEG chains
338
on the periphery of the conjugates blocked the drug cut from hydrazone bound to
339
penetrate. The findings agreed with those reported by Shen [6] who had verified that
340
PEG layer had certain effect on the drug release. Due to the molecular structural
341
characteristics of PAMAM and solubility of DOX, we presumed that part of the
342
released DOX from FPP-hyd-DOX was potentially re-encapsulated into the
343
hydrophobic core of PAMAM dendrimer.
344
3.3 In Vitro Cytotoxicity Studies
M
an
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334
The cytotoxicity of free DOX, blank carrier and PAMAM-DOX conjugates
346
against KB cells were studied using the MTT assay. As shown in Fig. 4A, all the
347
FPP-hyd-DOX exhibited higher cytotoxicity than PP-hyd-DOX, and the cell viability
349 350 351
te
Ac ce p
348
d
345
decreased with the number of FA ligands increasing, indicating that FA promoted the uptake of conjugates into the FR-overexpressed cancer cells and thus increased the intracellular drug accumulation. FPP-hyd-DOX 16/1 was determined to be the optimal formulation with the highest uptake and cytotoxicity, thus was selected to do
352
the following experiments. The IC50 value of FPP-hyd-DOX 16/1 (2.77 µM) was
353
2.2-fold lower than that of non-targeted conjugate PP-hyd-DOX (6.18 µM) (Fig. 4 C).
354
The viability of KB cells after 48 h incubation with blank carriers was shown in Fig.
355
4B. PAMAM dendrimers showed significant cytotoxicity against KB cells with an 17
Page 17 of 34
IC50 value of 2.33 μM. Introduction of FA-PEG-NHS reduced the cytotoxicity of
357
PAMAM, and more than half of the cells were still alive even at the highest
358
concentration (100 μM).
359
3.4 Cellular Uptake of PAMAM-DOX conjugates
ip t
356
To evaluate the effect of FA on targeting capacity, fluorescence microscopy was
361
used to study the cellular uptake characteristics. The results were shown in Fig. 5A-D,
362
as the incubation time increased, both the fluorescence intensity of KB cells treated
363
with FPP-hyd-DOX 16/1 and PP-hyd-DOX increased. At either 1 h or 2 h, the
364
fluorescence intensity of KB cells treated with FPP-hyd-DOX 16/1 was obviously
365
higher than that treated with PP-hyd-DOX.
M
an
us
cr
360
The quantitative analysis of cellular uptake was further investigated using FCM.
367
Flow cytometry histograms of cell-associated DOX fluorescence against KB cells
368
were shown in Fig. 5E-F. With the same incubation time and DOX concentration,
369
FPP-hyd-DOX 16/1 showed higher fluorescence intensity than free DOX and
371 372 373
te
Ac ce p
370
d
366
PP-hyd-DOX. After 2 h incubation, the FPP-hyd-DOX 16/1 showed a 1.4-fold increase in cellular uptake than that of PP-hyd-DOX. Free DOX was found to be similar in uptake with PP-hyd-DOX. These results suggested that receptor-mediated endocytosis played important role in the cellular uptake and FA ligands attaching on
374
the surface of conjugates significantly promoted the uptake [37].
375
3.5 Route of Cellular Uptake
376
Various uptake inhibitors, incuding Chlorpromazine, colchicines, filipin, PLL
377
and FA [37-40], were used to study the uptake route of PAMAM-DOX conjugates by 18
Page 18 of 34
KB cells. Many of previous reports have investigated the cellular internalization
379
mechanism of PAMAM dendrimers. The obtained results seemed to be dependent on
380
the cell type, the surface modification and particle size [15, 40, 41]. As shown in Fig.
381
6A, each inhibitor displayed no obvious cytotoxicity against KB cells. Fig. 6B
382
presented the cell uptake of PAMAM-DOX conjugates in the presence of various
383
inhibitors. Chlorpromazine decreased the cellular uptake of FPP-hyd-DOX 16/1 and
384
PP-hyd-DOX to 63.8% and 66.6% of the control, respectively, indicating that
385
clathrin-mediated endocytosis was blocked by disturbing the formation of
386
clathrin-coated pits [26, 30, 36]. Incubation with PLL reduced the cellular uptake of
387
FPP-hyd-DOX 16/1 and PP-hyd-DOX to 76.8% and 73.2% of the control,
388
respectively, displaying the importance of surface positive charge on the
389
internalization of PAMAM-DOX conjugates [39, 42, 43]. The effect of
390
caveolae-mediated endocytosis on the internalization of the conjugates was evaluated
391
using filipin, and no significant uptake difference was observed compared with each
393 394 395
cr
us
an
M
d
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Ac ce p
392
ip t
378
control. The minimal effect of colchicines on conjugates uptake showed that macro-pinocytosis was presumably involved to a lesser extent. Free FA decreased cellular uptake of FPP-hyd-DOX 16/1 to 82.4%, but had no effect on PP-hyd-DOX uptake, suggesting that FA-FR recognition was involved in the cellular uptake of
396
FPP-hyd-DOX 16/1 by FR overexpressing cells. Endocytosis inhibition experiments
397
demonstrated that PAMAM-DOX conjugates were internalized by KB cells mainly
398
through FR and clathria co-mediated endocytosis, while the contribution of
399
caveolae-mediated endocytosis and macro-pinocytosis was minimal. 19
Page 19 of 34
400
3.6 Subcellular Localization of the PAMAM-DOX Conjugates The subcellular localization of the conjugates in KB cells was evaluated by
402
confocal laser scanning microscopy (CLSM). As shown in Fig. 6C, free DOX, the red
403
fluorescence, was found to localize in the nuclei of KB cells, showing a
404
co-localization with the blue fluorescence of Hoechst 33342 (Fig. 6C a-d). As to
405
FPP-hyd-DOX 16/1 and PP-hyd-DOX, DOX related red fluorescence could be
406
observed both in the lysosomes and nuclei, displaying co-localization with
407
LysoTracker green (a specific marker for lysosome) and Hoechst 33342 (Fig. 6C e-h
408
and Fig. 6C i-l). The internalization of the FPP-hyd-DOX 16/1 was much higher than
409
that of PP-hyd-DOX. Chloroquine was applied to enhance the pH value of the
410
lysosome compartment of KB cells. As a result, no red fluorescence could be
411
observed in the nuclei, indicating that the acidic condition was essential for DOX
412
release from FPP-hyd-DOX 16/1 (Fig. 6C m-p). Confocal images showed that the
413
conjugates were delivered to lysosome where the slightly acidic conditions triggered
415 416 417
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the release of DOX, and then the released DOX was transported into the nuclei to exert anti-tumor effects [35]. 4 Conclusions
In this study, acid-labile PAMAM-DOX conjugates with different number of FA
418
ligands were successfully synthesized. Both in vitro drug release assay and CLSM
419
images exhibited that DOX release from the PAMAM-DOX conjugates depended
420
strongly on the pH values. As in lysosomes (pH4.5~6.5), DOX could be fast released
421
from the conjugates. Compared with non-targeted PP-hyd-DOX, FPP-hyd-DOX 16/1 20
Page 20 of 34
had obviously greater cytotoxicity and cellular uptake against KB cells, due to the
423
FR-mediated active targeting. Endocytosis mechanism studies further revealed that
424
the PAMAM-DOX conjugates were internalized by KB cells mainly through
425
clathrin-mediated endocytosis, and transported to lysosomes, where DOX was
426
released from the conjugates and subsequently entered into the nucleus. Overall, the
427
present study demonstrates that the FA-modified PAMAM-DOX conjugates possess
428
excellent targeting ability and intracellular pH-responsive drug release property.
429
Thereby, it may be a promising drug delivery carrier for targeted cancer therapy.
430
Acknowledgements
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The work was supported by National Natural Science Foundation of China
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(81173004 and 81302719) and PAPD (A Project Funded by the Priority Academic
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Program Developments of Jiangsu Higher Education Institutions, China).
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510 511 512
Number of
Feed ratio
Number of
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Table 1 Characteristics of PAMAM-DOX conjugates Particle size
Zeta potential
FA
FA:PAMAM
DOX
1
( H NMR)
(by UV-vis)
PAMAM
-
-
-
PP-hyd-DOX
-
-
FPP-hyd-DOX 4/1
4:1
3.3
FPP-hyd-DOX 8/1
8:1
5.8
FPP-hyd-DOX 16/1
16:1
10.1
an
(mv) (n=3)
(by volume)
us
(by mol)
(nm) (n=3)
cr
Conjugates
5.1 ± 0.1
18.90 ± 0.11
20.1 ± 0.3
4.53 ± 0.03
25.3 ± 0.2
4.79 ± 0.13
23.4 ± 0.3
4.67 ± 0.07
30.3 ± 0.9
4.88 ± 0.05
d te
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513
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10.1
26
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Graphical Abstract (for review)
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Scheme 1
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