Surface engineering of nanomaterials with phospholipid-polyethylene glycol-derived functional conjugates for molecular imaging and targeted therapy

Journal Pre-proof Surface engineering of nanomaterials with phospholipid-polyethylene glycolderived functional conjugates for molecular imaging and targeted therapy

Dinglin Zhang, Jianxiang Zhang PII:

S0142-9612(19)30745-8

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119646

Reference:

JBMT 119646

To appear in:

Biomaterials

Received Date:

25 April 2019

Accepted Date:

21 November 2019

Please cite this article as: Dinglin Zhang, Jianxiang Zhang, Surface engineering of nanomaterials with phospholipid-polyethylene glycol-derived functional conjugates for molecular imaging and targeted therapy, Biomaterials (2019), https://doi.org/10.1016/j.biomaterials.2019.119646

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier.

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Surface engineering of nanomaterials with phospholipid-polyethylene glycol-derived functional conjugates for molecular imaging and targeted therapy

Dinglin Zhang a,b,c,d,*, Jianxiang Zhang b,d,*

a Department

of Chemistry, College of Basic Medicine, Third Military Medical University (Amy Medical University),

Chongqing 400038, China b Department

of Pharmaceutics, College of Pharmacy, Third Military Medical University (Amy Medical University),

Chongqing 400038, China c Department

of Urology, Southwest Hospital, Third Military Medical University (Amy Medical University), Chongqing

400038, China d Chongqing

Key Laboratory for Disease Proteomics, Southwest Hospital, Third Military Medical University (Army

Medical University, Chongqing 400038, China

*Corresponding

authors:

Dinglin Zhang, PhD, Prof. Department of Chemistry College of Basic Medicine Third Military Medical University, Chongqing 400038, China E-mail: [email protected]; [email protected] ORCID: 0000-0003-4400-919X

Jianxiang Zhang, PhD, Prof. Department of Pharmaceutics College of Pharmacy Third Military Medical University, Chongqing 400038, China E-mail: [email protected]; [email protected] ORCID: 0000-0002-0984-2947

1

ABSTRACT

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In recent years, phospholipid-polyethylene glycol-derived functional conjugates have been widely employed to decorate different nanomaterials, due to their excellent biocompatibility, long blood circulation characteristics, and specific targeting capability. Numerous in vivo studies have demonstrated that nanomedicines peripherally engineered with phospholipid-polyethylene glycol-derived functional conjugates show significantly increased selective and efficient internalization by target cells/tissues. Targeting moieties including small-molecule ligands, peptides, proteins, and antibodies are generally conjugated onto PEGylated phospholipids to decorate liposomes, micelles, hybrid nanoparticles, nanocomplexes, and nanoemulsions for targeted delivery of diagnostic and therapeutic agents to diseased sites. In this review, the synthesis methods of phospholipid-polyethylene glycol-derived functional conjugates, biophysicochemical properties of nanomedicines decorated with these conjugates, factors dominating their targeting efficiency, as well as their applications for in vivo molecular imaging and targeted therapy were summarized and discussed.

Keywords Phospholipid-polyethylene glycol Targeting ligands Nanodiagnostics Nanotherapies Molecular imaging Targeted therapy

2

Abbreviations ADM AMB BODIPY CDI CEA CisPt CPT CT DCC DCU DiD DIPEA DiR DMAP DMF DMSO DMPE-PEG DOX DOPE-PEG DPPE-PEG DSPE-PEG DTX EDC EGFR FDA 5-Fu HER2 HRP ICG Mal MALDI-TOF MMC MR MS MTX NHS NMR ODNs oxSWNHs PCL PLGA PTX rMETase

Journal Pre-proof Adriamycin Amphotericin B Boron-dipyrromethene N,N'-Carbonyldiimidazole Carcinoembryonic antigen Cisplatin Camptothecin Computed tomography N,N’-Dicyclohexylcarbodiimide N,N’-Dicyclohexylurea 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine N,N-Diisopropylethylamine 1, 1’-Dioctadecyltetramethyl-3,3,3’,3’-indotricarbocyanineiodide 4-Dimethylamino pyridine N,N-Dimethylformamide Dimethyl sulfoxide 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)) Doxorubicin 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine-N-(methoxy(polyethylene glycol)) 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) Docetaxel 1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide Epidermal growth factor receptor Food and Drug Administration 5-Fluorouracil Human epidermal growth factor receptor 2 Horse-radish peroxidase Indocyanine green Maleimide Matrix-assisted laser desorption/ionization time-of-flight Mitomycin C Magnetic resonance Mass spectrometry Methotrexate N-Hydroxysuccinimide Nuclear magnetic resonance Oligodeoxyribonucleotide Oxidized single-wall carbon nanohorns Polycaprolactone Poly(lactic-co-glycolic acid) Paclitaxel Recombinant methioninase 3

SA SAL TEA US WSBA 90Y

Journal Succinic anhydride Salinomycin Triethylamine Ultrasonic sound Water-soluble boronated acridine-1 Yttrium-90

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4

1. Introduction

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Nanotechnology has rapidly developed and innovated in the past few decades, which has been extensively employed to serve biology, biomedical engineering, pharmaceutics, and medicine [1]. Precision diagnosis and therapy using functional nanoparticles (NPs) is one of the most active research fields of nanotechnology. In this aspect, numerous imaging agents and drugs with different physicochemical properties have been packaged into NPs for diagnosis and treatment of a plethora of diseases, such as cancer [2], inflammatory bowel disease [3], cardiovascular disease [4], and diabetes [5]. For example, various nanoprobes have been explored for near-infrared fluorescence imaging [6], computed tomography [7], magnetic resonance imaging [8], ultrasound imaging [9], etc. On the other hand, a large number of nanotherapeutics have been developed to achieve targeted delivery of therapeutic molecules, including small-molecule drugs, nucleic acids, antibodies, peptides, proteins, among others [1]. Compared to traditional drugs, nanomedicines have multiple advantages such as decreased side effects, reduced dosing frequency, improved pharmaceutical properties, and enhanced targeting capability [10]. Both diagnostic and therapeutic cargos can be site-specifically delivered to diseased sites via passive and/or active targeting strategies. Since passive targeting is mainly based on the enhanced permeability and retention (EPR) effect in most cases [11, 12], it is notably affected by physicochemical properties of NPs, such as particle size, size distribution, surface chemistry, and particle shape [13, 14]. To enhance targeting efficiency by prolonging the blood circulation time of NPs, surface coating of NPs with polyethylene glycol (PEG) chains is a commonly used strategy [14]. In addition, PEGylation can decrease the immunogenicity of NPs. Some PEGylated therapeutics have been approved by FDA for the treatment of rheumatoid arthritis and age-related macular degeneration [15]. Although PEGylation of NPs can enhance their EPR effect, passive targeting is often limited by the lack of specificity to target sites [16]. Alternatively, based on the overexpression of specific molecules on the surface of disease-related cells (e.g., inflammatory cells and cancer cells), active targeting provides an effective way to increase accumulation of NPs at diseased sites [17, 18]. In this approach, targeting moieties need to be introduced on the surface of NPs to formulate nanomedicines with active targeting capability. PEGylated phospholipids are conjugates of phospholipids and PEG which have been widely used for construction of different drug delivery systems [19, 20]. PEGylated phospholipid-based nanocarriers or NPs coated with PEGylated phospholipids can, to a certain degree, escape from the reticuloendothelial system, thereby having long blood circulation time. Among these PEGylated phospholipids, DSPE-PEG is the most widely used one, due to its capability of more significantly enhancing circulating time in vivo than other PEGylated phospholipids [21]. Targeting moieties can be easily conjugated onto the distal end of PEG in phospholipid-PEG conjugates via various chemical coupling strategies. As the targeting components, phospholipid-PEG-ligand conjugates have multiple merits: 1) The conjugation reaction of PEGylated phospholipids and targeting moieties generally can be performed by one step coupling reaction under mild 5

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reaction conditions; 2) Phospholipid-PEG-ligand conjugates themselves can serve as self-assembling nanocarriers; 3) Insertion of targeting moieties on the surface of PEGylated phospholipid-decorated NPs does not significantly alter their physicochemical properties such as polydispersity index (PDI), drug loading capacity, and encapsulation efficiency (EE), while the targeting efficiency can be enhanced several folds compared to non-targeted counterparts. Therefore, phospholipid-PEG-ligand conjugates are extensively used to modify various nanovehicles including liposomes, micelles, hybrid NPs, nanocomplexes, and nanoemulsions (Fig. 1). Whereas some phospholipid-PEG-ligand conjugates are commercially available due to their broad development prospect, much more functional phospholipid-PEG conjugates need to be synthesized case-by-case for molecular imaging and targeted therapy. Unfortunately, currently there is no available literature that comprehensively reviews the structure, synthesis, and nanomedicine applications of different phospholipid-PEG-ligand conjugates. In this review, we aim to provide comprehensive knowledge regarding the molecular structures and synthesis methods of different phospholipid-PEG conjugates, as well as the physicochemical properties and factors dominating targeting efficiency of nanomedicines formulated using phospholipid-PEG conjugates. Furthermore, we thoroughly discussed the current progress in molecular imaging and targeted therapy of diverse diseases using nanomedicines peripherally decorated with phospholipid-PEG-derived functional conjugates.

2. Synthesis of diverse phospholipid-PEG-derived functional conjugates Various chemical coupling strategies (e.g., thiol-maleimide coupling strategy, EDC/NHS coupling chemistry, and click chemistry approach) have been employed to conjugate targeting moieties onto phospholipid-PEG materials to afford functional carries. In order to facilitate the coupling reaction, reactive functional groups (i.e., amino, carboxyl, sulfhydryl, and maleimide groups) are generally introduced to carrier materials or targeting moieties. Since there are already some reviews that summarize the category of targeting moieties, linkers, and conjugation methods [18, 22-24], herein we mainly focus on the synthesis of phospholipid-PEG-based targeting conjugates.

2.1. Functionalization of PEGylated phospholipids and different targeting moieties PEGylated phospholipids (e.g., DSPE-PEG, DPPE-PEG, and DOPE-PEG) with various PEG chain lengths (i.e., PEG550, PEG1000, PEG2000, PEG3350, and PEG5000) have been used for decoration of NPs. For these PEGylated phospholipids, PEGs with molecular weight of 2000 (PEG2000) or greater are necessary to ensure long blood circulation for resulting NPs [25]. To conjugate targeting molecules, fluorescence probes, or therapeutics onto the PEG chains, various functional groups such as amino, carboxyl, and maleimide are frequently introduced to the distal end of PEG. All these functionalized PEGylated phospholipids can be synthesized in a laboratory or commercially obtained due to their broad applications in pharmaceutics 6

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and biomedical engineering. Among these functionalized PEGylated phospholipids, DSPE-PEG-Mal, DSPE-PEG-NHS, and DSPE-PEG-NH2 are used more broadly than others due to the excellent long blood circulation characteristics of DSPE-PEG-decorated NPs and high reaction activity of the corresponding functional groups. The synthesis methods for different functionalized PEGylated phospholipids are summarized in Fig. 2A. For instance, DSPE-PEG-COOH can be synthesized through conjugation of DSPE and PEG-bis(succinimidyl succinate) with triethylamine as a catalyst (Fig. 2A(i)) [26-28]. The purified DSPE-PEG-COOH can be obtained by separation through thin-layer chromatography. Using EDC as activator and NHS as a reactant, DSPE-PEG-COOH can be derived to DSEP-PEG-NHS. In addition, by protection of the amino of PEG with butyloxycarbonyl (Boc), the NHS-activated carboxyl group at the other end of PEG can react with DSPE to synthesize DSPE-PEG-NH-Boc [29]. The protective group of DSPE-PEG-NH-Boc can be then removed in HCl/dioxane solution to yield DSPE-PEG-NH2 (Fig. 2A (ii)). Otherwise, DSPE-PEG-NH2 can be prepared through reaction of NH2-PEG-NH2 with NHS-activated DSPE-succinic acid [30]. Subsequently, DSPE-PEG-NH2 can further react with N-succinimidyl-3-(N-maleimido)-propionate to prepare DSPE-PEG-Mal (Fig. 2A (iii)) [31]. Moreover, DSPE-PEG-Mal can also be synthesized through conjugating ω-(β-[N-maleimido])-PEG-α-succinimidyl carboxylate (Mal-PEG-SC) onto DSPE (Fig. 2A (iv)). Targeting units with reactive functional groups such as amino and carboxyl groups can be directly conjugated onto carriers via amidation or esterification reaction. By using organic alkalines (e.g., TEA or DMAP) and coupling reagents (i.e., CDI or EDC), the coupling reaction can be accelerated. To improve reactivity of targeting moieties, sulfhydryl or NHS is frequently employed to activate them. To introduce the sulfhydryl group onto a targeting unit, its amino group can be activated by 2-iminothiolane [32, 33] or N-succinimidyl-S-acethylthioacetate (SATA) [34]. Alternatively, the redox reaction is applied to cleave disulphide bonds of targeting moieties to obtain the native sulfhydryl group [35-37]. Otherwise, the carboxyl group of targeting units can be activated by EDC/NHS or DCC/NHS [38].

2.2. Methods for covalent conjugation of targeting moieties onto PEGylated phospholipids The thiol-maleimide coupling strategy, EDC/NHS coupling chemistry, and amidation reaction are frequently used to conjugate targeting units onto PEGylated phospholipids (Fig. 2B). 2.2.1. The thiol-maleimide coupling strategy The thiol-maleimide coupling strategy is the most frequently used approach to conjugate different targeting moieties onto carrier materials. Targeting moieties containing the sulfhydryl group can react easily with maleimide-modified PEGylated phospholipids via thiol-maleimide coupling reaction under extremely mild conditions. The conjugation reaction can be performed in aqueous solution at pH 7.0-7.5 [39], with high selectivity and yield. 2.2.2. DCC/NHS or EDC/NHS coupling chemistry 7

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DCC/NHS or EDC/NHS coupling chemistry is broadly used in organic synthesis, peptide synthesis, bioconjugation, and immunochemistry [40]. DCC/NHS-mediated coupling chemistry has been early employed to conjugate carboxyl with amino. However, it is generally very difficult to separate the byproduct of DCC from the desired product. Accordingly, DCC/NHS coupling chemistry has been replaced by EDC/NHS coupling chemistry in most cases, due to the good water-solubility of the EDC-derived byproduct. When the carboxyl group of a targeting moiety is activated by EDC/NHS, it reacts easily with phospholipid-PEG-NH2. Similarly, phospholipid-PEG-COOH can be activated by EDC/NHS, and then it can react with the amino group of a targeting unit. Generally, EDC/NHS coupling reaction is rapid in a weak acid solution (pH 6.0-6.5) [40]. Nevertheless, the conjugation reaction of targeting moieties and PEGylated phospholipids was performed in alkaline solutions (pH 7.5-10.0) in some cases [41]. 2.2.3. The direct amidation strategy The amide bond can be formed through the condensation reaction of amino and carboxyl, in which an organic alkaline (e.g., DMAP, TEA, or pyridine) and a condensation agent (i.e., DCC or EDC) are frequently used to facilitate the reaction. Raising the reaction temperature is beneficial for the condensation reaction. However, the byproduct of catalysts (e.g., DCU) cannot be easily separated from the final product even by dialysis, precipitation, or column chromatography methods. Therefore, this coupling strategy is suitable for conjugation of small molecule targeting moieties onto PEGylated phospholipids. In addition to the above mentioned methods, click chemistry [42, 43], redox reaction[44], and cyanuric chloride linking [45, 46] are also employed to conjugate targeting units onto PEGylated phospholipids.

2.3. Synthesis of PEGylated phospholipid conjugates with different targeting moieties In the past decades, various targeting moieties (e.g., small molecule ligands, peptides, proteins, antibodies, and aptamers) have been conjugated onto PEGylated phospholipids to synthesize phospholipid-PEG-derived targeting conjugates. Both pre-conjugation and post-insertion strategies are generally employed to prepare functional phospholipid-PEG conjugates. For the pre-conjugation method, the conjugation of targeting moieties with PEGylated phospholipids is relatively straightforward and performed before the preparation of NPs. By contrast, phospholipid-PEG-linkers are first used to fabricate NPs, and then targeting moieties are attached onto the surface of NPs via chemical coupling in the post-insertion method. The functional conjugates of PEGylated phospholipids with various targeting moieties synthesized via directly coupling can be purified by dialysis or column chromatography methods, with their structures can be characterized by 1H NMR spectroscopy and MALDI-TOF MS. On the other hand, NPs peripherally modified with phospholipid-PEG targeting conjugates via the post-insertion method can be purified via centrifugation. The amount of phospholipid-PEG conjugates on NPs can be calculated by subtracting the amount of targeting conjugates detected in the filtrate from that of the initially 8

used.

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2.3.1. Synthesis of PEGylated phospholipid targeting conjugates with small molecule ligands As targeting moieties, small molecule ligands have multiple advantages, such as good stability, easy modification, and broad availability. Consequently, small molecule ligands (e.g., folic acid, methotrexate, anisamide, cholic acid, daptomycin, and sugars) have been conjugated onto PEGylated phospholipids to decorate various NPs. Among these ligands, folic acid (FA) draws much more attention than others. As well known, FA-modified NPs can target the delivered cargos to both inflammatory and cancer sites, due to the overexpression of the FA receptor (FR) [47]. Since FA has two reactive functional groups, i.e.carboxyl and amino groups, it can be easily conjugated onto PEGlated phospholipids. Several strategies have been employed to synthesize phospholipid-PEG-FA conjugates. Direct amidation is frequently performed to conjugate FA onto PEGylated-phospholipids [48-50]. In this strategy, the condensation agents (i.e., DCC and EDC) and alkalamides (e.g., DMAP or TEA) must be used for efficient conjugation (Fig. 3A). In addition, phospholipid-PEG-FA conjugates can be synthesized via coupling of phospholipid-SA and NH2-PEG-NH2 [51]. In this method, FA is first conjugated onto NH2-PEG-NH2 with DCC as a catalyst to form FA-PEG-NH2. In parallel, phospholipids can react with succinic anhydride to synthesize phospholipid-SA. Subsequently, phospholipid-PEG-FA conjugates can be obtained through coupling reaction between FA-PEG-NH2 and phospholipid-SA (Fig. 3B). The thiol-maleimide coupling strategy is another approach to synthesize phospholipid-PEG-FA. In this case, FA is activated by DCC/NHS firstly, followed by reaction with cysteamine to form FA-SH. FA-SH containing the sulfhydryl group can react easily with phospholipid-PEG-Mal via thiol-maleimide coupling chemistry to afford phospholipid-PEG-FA conjugates (Fig. 3C) [52]. Alternatively, the sulfhydryl group can be introduced to FA through reaction with glutathione (Fig. 3D) [53]. DCC/NHS or EDC/NHS coupling chemistry is also extensively employed to conjugate phospholipid-PEG-NH2 and NHS-activated FA to afford phospholipid-PEG-FA conjugates (Fig. 3E) [54, 55]. These reactions are often performed in hydrophilic organic solvents (e.g., DMSO and DMF) due to the excellent solubility of PEGylated phospholipids and FA in these solvents. The post-insertion method has been also developed to fabricate phospholipid-PEG-FA modified liposomes [56]. In this aspect, 6-maleimidocaproic acid is conjugated onto phospholipids with DCC as an activator to form phospholipid-Mal, thereby fabricating phospholipid-Mal-modified liposomes. On the other hand, NH2-PEG-NH2 is thiolated through reaction with 2-iminothiolane to obtain NH2-PEG-SH. Phospholipid-Mal modified liposomes are PEGylated by NH2-PEG-SH via thiol-maleimide coupling. Finally, phospholipid-PEG-FA modified liposomes are obtained via conjugating NHS-activated FA onto liposomes modified with phospholipid-PEG-NH2 (Fig. 3F). Sigma receptors are nonopioid, nondopaminergic, membrane-bound proteins that are overexpressed in a variety of human tumor cells, including malignant melanoma, non-small cell lung carcinoma, breast tumors of neural origin, and prostate cancer cells [57]. Benzamides are applied to treat sigma receptor-overexpressed tumors due to its high affinity to 9

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sigma receptors. As a category of benzamides, anisamide (AA) is conjugated to DSPE-PEG-linker to form DSPE-PEG-AA conjugate. For example, N-(2-bromoethyl)-4-methoxy-benzamide was conjugated onto DSPE-PEG-NH2 in acetonitrile with DIPEA as a catalyst to synthesize DSPE-PEG-AA conjugate [58]. DSPE-PEG-AA conjugate has been widely used to decorate various NPs which are employed to deliver therapeutics and diagnostics to treat cancers with overexpressed sigma receptors. Similarly, EDC/NHS coupling chemistry is adopted to conjugate methotrexate [59, 60], biotin [38], sialyl LewisX [61], mannosyl [62], alendronate [63], or daptomycin [64] onto PEGylated phospholipids. Cholic acid can be directly conjugated onto DSPE-PEG-NH2 via an amidation strategy with DCC as a catalyst [65, 66]. In other cases, esterification reaction is explored to synthesize phospholipid-PEG-riboflavin conjugate [67]. 2.3.2. Synthesis of PEGylated phospholipids conjugated with peptides/proteins Peptides are widely used as targeting moieties, owning to their stable structure, low toxicity, small size, and reactive functional groups. Either amino or carboxyl in peptides and proteins can be conjugated with carrier materials via amidation coupling. In addition, some peptides and proteins possessing the sulfhydryl group can be conjugated onto carriers through thiol-maleimide coupling chemistry. RGD (Arg-Gly-Asp) is a tripeptide that is able to selectively recognize integrin (i.e., αvβ3, αvβ5, and α5β1) [68]. Due to the specific affinity of RGD and integrin receptors, cellular uptake of RGD-decorated nanovehicles by integrin-overexpressing cells can be remarkably enhanced via integrin receptor-mediated endocytosis [69]. Thiolated-RGD peptides can be easily conjugated onto DSPE-PEG-Mal through thiol-maleimide coupling in buffer solution (pH 7.0-8.0) at room temperature (Fig. 4A) [70, 71]. EDC/NHS coupling chemistry is another important pathway to conjugate RGD peptides onto DSPE-PEG-COOH (Fig. 4B) [72-75]. In addition, click chemistry reactions are explored to conjugate hexynoic acid-modified RGD peptide onto DSPE-PEG-N3 to prepare DSPE-PEG-RGD conjugate (Fig. 4C) [42, 76]. On the other hand, nanovehicles modified with phospholipid-PEG-RGD conjugates can be fabricated by a post-insertion method (Fig. 4D). For example, DSPE-PEG-COOH modified nanocapsules were first activated by EDC/NHS, followed by reaction with RGD to form RGD-coated nanocapsules that were further purified by ultracentrifugation. Also, RGD peptides can be attached onto DSPE-PEG-NH2 decorated liposomes using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a catalyst (Fig. 4E) [77]. In addition to RGD peptides, other peptides and proteins are conjugated onto PEGylated phospholipids to prepare phospholipid-PEG-peptide/protein conjugates (Fig. 5). In these cases, EDC/NHS and thiol-maleimide coupling chemistries are still the most frequently used methods to synthesize phospholipid-PEG-peptide/protein conjugates. For example, EDC/NHS coupling was employed to conjugate luteinizing hormone-releasing hormone peptide [78], transferrin receptor targeting peptide [79, 80], bradykinin [81, 82], chlorotoxin [83], transferring [84-86], or lactoferrin [87] onto PEGylated 10

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phospholipids. Cell penetrating peptides [88-97], EGFR targeting peptides [98, 99], antagonist G [32, 100], P-selectin [101], and fibronectin extra domain B-specific aptide [102-104] were conjugated onto PEGylated-phospholipids through thiol-maleimide coupling chemistry. Alternatively, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and succinimidyl propionate (SPA) were used to functionalize PEGylated phospholipids to introduce a disulfide bond, and then trans-activation transcription peptide or vasoactive intestinal peptide was conjugated onto PEGylated phospholipids via a disulfide linkage [105, 106]. Cyanuric chloride was also explored as a linker to couple ephrin-A1 protein with PEGylated phospholipids [46]. 2.3.3. Synthesis of PEGylated phospholipids conjugated with antibodies or aptamers Antibodies have also been widely used as targeting moieties due to their high affinities and specific recognition capabilities to their targets. For antibodies, their amino groups can react with EDC/NHS-activated or cyanuric chloride-linked PEGylated phospholipids via EDC/NHS coupling chemistry (Fig. 6A) [107] and nucleophilic substitution reaction (Fig. 6B) [45], respectively. Alternatively, N-succinimidyl-S-acethylthioacetate (SATA)-modified antibodies can be conjugated onto maleimide-activated PEGylated phospholipids through thiol-maleimide coupling chemistry (Fig. 6C) [34]. Compared to peptides and small-molecule ligands, antibodies have bigger size and more complicated structure. Using the pre-conjugation method, it is difficult to ensure efficient coating of antibodies on the surface of nanovehicles. Therefore, post-insertion is an optimal pathway to fabricate nanovehicles modified with phospholipid-PEG-antibody conjugates (Fig. 6D-J). SPDP (Fig. 6D) [108], 2-iminothiolane (Fig. 6E), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Fig. 6I) [37, 109], 2-mercaptoethylamine [110], and 3-(2-pyridyldithio)-propionyl hydrazide (PDPH) (Fig. 6J) [44] are widely utilized to thiolate antibodies and antibody fragments. The thiolated antibodies can be attached onto DSPE-PEG-Mal-modified nanovehicles via thiol-maleimide coupling. Otherwise, the maleimide group can be introduced into antibodies through reaction with N-succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB) (Fig. 6H). In this case, the maleimide-modified antibody is conjugated onto DSPE-PEG-SH-modified liposomes via a post-insertion method [111]. Additionally, DSPE-PEG-NH2-decorated NPs can be activated by bis(sulfosuccinimidyl) suberate (BS3). Antibodies are then attached onto DSPE-PEG-NH2-modified NPs after BS3 activation (Fig. 6F) [38]. EDC/NHS coupling chemistry can be also used in the post-insertion strategy to fabricate nanovehicles modified with phospholipid-PEG-antibody conjugates. For instance, DSPE-PEG-COOH-modified liposomes were first activated by EDC/NHS, and then antibodies were attached onto DSPE-PEG-NHS-modified liposomes through post-insertion (Fig. 6G) [28, 112, 113]. Aptamers, a class of oligonucleotide or peptide molecules with specific binding capability to target molecules, can be also conjugated onto PEGylated phospholipids. For example, aptamer derivatives containing disulfide bond can be reduced by TCEP to obtain thiolated aptamers. The thiolated aptamers then react with DSPE-PEG-Mal to form DSPE-PEG-aptamer conjugates [35, 36, 114]. Alternatively, aptamers can be directly conjugated onto EDC/NHS-activated DSPE-PEG-COOH 11

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via EDC/NHS coupling chemistry [115]. Taken

together,

benefited

from

the

tremendous

progress

of

organic

synthesis

methods,

multifarious

PEG-phospholipid-derived functional conjugates have been synthesized with high selectivity and perfect yield. Among these synthesis methods, EDC/NHS and thiol-maleimide coupling chemistries are the most desirable approaches for synthesis of functional PEG-phospholipid conjugates, because these coupling reactions can be completed under mild conditions that is beneficial to protecting the chemical structure ofPEGylated phospholipids and targeting moieties. Whereas other click chemistry reactions can also be performed under mild reaction conditions, the relatively complicated procedures and high cost largely limit its wide applications. Small-molecule ligands are stable, and their reactive functional groups can be easily conjugated with PEGylated phospholipids. Conjugates of phospholipid-PEG and small-molecule ligands have been widely used to decorate different nanovehicles (e.g., liposomes, micelles, hybrid NPs, and nanoemulsions) for targeted cargo delivery to pathological sites. Nevertheless, these nanovehicles are largely used for diagnosis and therapy of cancers and inflammation-associated diseases. PEGylated phospholipids functionalized with peptides or antibodies are broadly explored to achieve site-specific delivery in various diseases, such as cancers, vascular diseases, and neurodegenerative diseases. Peptides and antibodies have fragile bonds such as disulphide and amide linkages. Therefore, the synthesis method must be carefully considered during preparation of phospholipid-PEG-peptide/antibody conjugates. It is noteworthy that fabrication by the pre-conjugation method cannot ensure all the targeting moieties at the periphery of NPs modified with phospholipid-PEG-ligand conjugates, mainly due to the flexibility of PEG chains. This issue can be addressed by the post-insertion method, although the related preparation process is more complex compared to that of the pre-conjugation method.

3. Surface engineering of nanocarriers using phospholipid-PEG-ligand conjugates and their targeting capability Phospholipid-PEG-ligand conjugates are extensively utilized to decorate liposomes, micelles, polymeric hybrid NPs, and inorganic hybrid NPs to afford long blood circulation and specific targeting characteristics for resulting nanovehicles. To a certain degree, decoration with phospholipid-PEG-ligand conjugates may affect the physicochemical characteristics of NPs, such as particle size, PDI, and surface charge, which play an important role in determining their in vivo targeting efficiency and efficacies [116]. Moreover, phospholipid-PEG linkers and the combinations of PEGylated phospholipids with phospholipid-PEG-ligand conjugates can also influence targeting efficiency of NPs. In the following sections, we describe different types of NPs decorated with phospholipid-PEG-ligand conjugates and the factors dominating their targeting efficiency.

3.1. Phospholipid-PEG-ligand conjugates for surface decoration of liposomes 12

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Liposomes are the most extensively investigated nanocarriers for biomedical applications [117]. Some drug-loaded liposomes such as Doxil, Daunoxome, and Marqibo have been approved by FDA for clinical use. The first generation liposomes are employed for passive targeting of different diseases through the EPR effect [118]. Prolonged blood circulation of liposomes is beneficial for their passive accumulation in pathological sites. To achieve this purpose, PEGylated phospholipids are frequently employed to decorate liposomes. For some tumors, however, the EPR effect only results in modest therapeutic efficacy due to poor tumor penetration, high interstitial pressure, and inefficient tumor cell uptake. To overcome this shortcoming, phospholipid-PEG-ligand conjugates are applied to modify liposomes to realize actively targeted drug delivery. In general, the pre-conjugation and post-insertion methods are used to fabricate targeting liposomes decorated with phospholipid-PEG-ligand conjugates. In the pre-conjugation method, phospholipid-PEG-ligand conjugates are first synthesized, and then liposomes are prepared by the conventional methods in the presence of phospholipid-PEG-ligand conjugates. It has been demonstrated that high contents of PEG (>15% in weight) may destroy the phospholipid bilayers [119]. Post-insertion is another frequently employed approach to fabricate phospholipid-PEG-ligand modified liposomes. In this method, liposomes are prepared firstly using functional phospholipid-PEG-linkers, such as DSPE-PEG-Mal, DSPE-PEG-NHS, DSPE-PEG-NH2, DPPE-PEG-Mal, and DOPE-PEG-Mal. Subsequently, liposomes decorated with phospholipid-PEG-linkers are dispersed in buffer solutions and modified by active targeting moieties via different coupling strategies. Santos et al. have proved that liposomes prepared by post-insertion exhibited 2-fold higher encapsulation efficiency for siRNA as compared to that based on the pre-conjugation method [100]. In addition, particle size and PDI of liposomes prepared by the pre-conjugation method were considerably larger than those derived from the post-insertion method (326 ± 135 nm versus 190 ± 34 nm). Oswald et al. also investigated liposomes obtained from these two preparation methods by using DSPE-PEG2000-Mal as a linker [39]. Similarly, liposomes with smaller mean size were obtained by the post-insertion method compared to those based on the pre-conjugation method (65 ± 3 nm versus 139 ± 5 nm). As for the two preparation methods mentioned above, targeting liposomes fabricated by the post-insertion method generally have the smaller size and higher drug loading contents, and therefore it is more promising strategy for practical applications.

3.2. Phospholipid-PEG-ligand conjugates for surface coating of polymeric and inorganic hybrid NPs By combining the advantages of polymeric NPs and liposomes, polymeric/lipid hybrid NPs have emerged as a promising delivery platform [120]. Generally, these hybrid NPs possess core-shell structure, with a polymeric core and a phospholipid shell. Cargos, such as small-molecule drugs, peptides/proteins, and siRNAs are loaded in the polymeric core of hybrid NPs. The lipid shell provides steric stabilization for the hybrid NPs. For some materials, it is difficult to form NPs by the emulsion-solvent evaporation or dialysis method, while core-shell hybrid NPs can be obtained by the 13

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nanoprecipitation/self-assembly method [121]. The hybrid architecture has multiple advantages such as desirable PDI, easy surface functionality, ideal drug loading, and good stability in blood circulation. Different methods, such as nanoprecipitation/self-assembly [122, 123], thin-film hydration/ultrasonic dispersion [124], nanoemulsion-solvent evaporation [125, 126], and emulsification/solvent diffusion [127] are usually employed to fabricate polymeric hybrid NPs decorated with phospholipid-PEG-ligand conjugates. Polyester (e.g., PLGA and PCL), polyethyleneimine (PEI), poly(amidoamine) (e.g., PAMAM), and polysaccharides are the frequently used materials to construct the core of polymeric hybrid NPs. The outer shell of hybrid NPs is composed of various phospholipids and phospholipid-PEG-ligand conjugates. The physicochemical properties of hybrid NPs are mainly dominated by the density of phospholipid conjugates, types of cargos, polymeric core, and fabrication methods. On the other hand, metal and inorganic NPs are extensively used for imaging as well as radiation and photothermal therapy of various tumors and other diseases. To enhance targeting capability of inorganic NPs, phospholipid-PEG-ligand conjugates are also explored to decorate the shell of these NPs. Nanoemulsion and thin-film rehydration methods are usually used to fabricate inorganic hybrid NPs coated with phospholipid-PEG-ligand conjugates. For the inorganic hybrid NPs, the core components can be magnetic resonance (MR) imaging contrast agents (e.g., gadolinium [128], superparamagnetic iron oxide NPs [104]) and fluorescent nanoprobes (such as quantum dots) [129] as well as drug-loaded polymeric NPs. In recent years, polymeric and/or inorganic core-shell NPs decorated with phospholipid-PEG-ligand conjugates have showed fantastic prospects owing to their desirable stability, good dispersibility, long blood circulation time, and active targeting capability. Inorganic NPs with uniform size, stable composition, and some special properties have been widely explored for diagnosis and therapy of various diseases. However, the potential toxicity and lack of specific distribution in vivo restrict the further clinical translation of inorganic NPs. Functionalization of inorganic NPs with phospholipid-PEG-ligand conjugates may provide an effective approach to overcome these limitations.

3.3. Ligand-conjugated PEGylated phospholipids for decoration of other nanovehicles Micelles are another promising nanoplatform for drug delivery due to their excellent biocompatibility, prolonged circulation, and in vivo degradability [130]. The hydrophobic core of micelles can efficiently load poorly water-soluble drugs, and their size can be tailored for passive accumulation at diseased sites. By modification with phospholipid-PEG-ligand conjugates, the active targeting efficiency of micelles can be considerably improved. Pre-conjugation is the most frequently used method for construction of micelles with phospholipid-PEG-ligand conjugates. In this approach, PEGylated phospholipids, phospholipid-PEG-ligand conjugates, and drugs are first dissolved in a hydrophilic organic solvent (i.e., DMSO or DMF), and thus obtained solution is dialyzed against deionized water to afford drug-loaded targeting micelles. Hydrophobic small-molecule drugs are usually loaded in micelles decorated with phospholipid-PEG-ligand conjugates. Of note, decoration with phospholipid-PEG-ligand conjugates showed no significant 14

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effects on the physicochemical properties of micelles, such as the particle size, PDI, drug loading content, and encapsulation efficiency, as compared to the non-targeted control micelles [90, 131-133]. In addition to the above mentioned nanovehicles, phospholipid-PEG-ligand conjugates are employed to decorate nanoemulsions [134], nanocapsules [135], carbon nanotubes [136], nanocages [137], and nanocomplexes [52, 138-143]. Nanoemulsions modified with phospholipid-PEG-ligand conjugates can be prepared by the injection method. For example, phospholipid-PEG-ligand conjugates, cholesterol, and drugs were first dissolved in a common organic solvent, which was removed by evaporation, resulting in the formation of semisolid solution. Subseqeuntly, 0.9% NaCl aqueous solution was injected to the semisolid solution to form nanoemulsions [134]. On the other hand, small-molecule drugs, RNAs, and DNAs can form nanocomplexes with phospholipid-PEG-ligand conjugates via self-assembly. The compositions of nanocomplexes are different from hybrid NPs, liposomes, and micelles.

3.4. Factors dominating targeting capability of phospholipid-PEG-ligand-decorated nanomedicines 3.4.1. Types of phospholipid-PEG linkers DSPE-PEG-Mal and DSPE-PEG-COOH are two broadly used linkers to conjugate targeting moieties. Using DSPE-PEG-Mal and DSPE-PEG-COOH as linkers, the post-insertion method was employed by Deng et al. to conjugate anti-EGFR Fab’ on the surface of liposomes to clarify its effect on targeting efficiency [144]. There were no significant differences in particle size, siRNA encapsulation efficiency, cell viability, and serum stability between the two liposomes. However, opposite ζ-potential was detected for liposomes based on the two linkers (8 mV for DSPE-PEG2000-Mal and -11 mV for DSPE-PEG2000-COOH). Cellular uptake of siRNA-loaded liposomes with DSPE-PEG2000-Mal as a linker was significantly higher (∼2 fold) than that of DSPE-PEG2000-COOH-based liposomes in EGFR-overexpressing SMMC-7721 cells. The luciferase gene silencing efficiency of siRNA-loaded liposomes based on DSPE-PEG-Mal was approximately 3-fold higher than that of DSPE-PEG2000-COOH-derived liposomes. These results indicated that DSPE-PEG2000-Mal is a more favorable linker to conjugate targeting units as compared to DSPE-PEG2000-COOH. 3.4.2. Pairs of PEGylated phospholipids and phospholipid-PEG-ligand conjugates Pairs of PEGylated phospholipids and phospholipid-PEG-ligand conjugates significantly affect targeting efficiency of nanovehicles. Human epidermal growth factor receptor 2 (HER2) is usally overexpressed on some breast cancer cells (such as BT-474 and SK-BR-3 cells). In order to investigate the influence of DSPE-PEG and DSPE-PEG-ligand on tumor cellular uptake, a short cyclic peptide antagonist of HER2 was used as the targeting ligand by Bilgicer et al., which was linked with DSPE-PEG to prepare DSPE-PEG-peptide conjugate [145]. In this study, cellular internalization experiments were conducted using nanovehicles formulated with DSPE-PEG-peptide (the length of PEG varied from 350, 500, 750, 1000, 1350, 1450, 2000, to 3350 Da) and DSPE-PEG350 pairs. The authors found that cellular uptake of 15

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DSPE-PEG3350-peptide/DSPE-PEG350 NPs and DSPE-PEG2000-peptide/DSPE-PEG350 NPs (both are targeting NPs) was not enhanced compared to the corresponding non-targeting NPs. By contrast, in comparison to NPs coated with DSEP-PEG350 conjugate alone, DSPE-PEG550-peptide/DSPE-PEG350 NPs and DSPE-PEG750-peptide/DSPE-PEG350 NPs showed dramatically enhanced cellular uptake by BT-474 and SK-BR-3 cells. For different targeting moieties and in varied cell types, the biological effects of PEGylated phospholipids paired with phospholipid-PEG-ligand conjugates might be different. For example, Saw et al. synthesized DSPE-PEG2000-APTEDB (APT, i.e., aptides that are a class of high-affinity peptides specific to extradomain B of fibronectin) and DSPE-PEG1000-APTEDB conjugates and paired them with DSPE-PEG with diverse molecular weights (PEG2000, PEG1000, PEG550, and PEG350) to fabricate targeting liposomes [102]. Cellular uptake assay

showed

that

liposomes

modified

with

DSPE-PEG2000-APTEDB/DSPE-PEG1000

or

DSPE-PEG1000-APTEDB/DSPE-PEG550 pairs had the highest uptake in U87MG cancer cells. Furthermore, in U87MG and SCC-7

xenograft

models,

liposomes

modified

with

DSPE-PEG2000-APTEDB/DSPE-PEG1000

or

DSPE-PEG1000-APTEDB/DSPE-PEG550 showed significantly higher accumulation in tumor sites than liposomes derived from other pairs. Accordingly, to achieve a most desirable targeting efficiency, the pairs of PEGylated phospholipids and phospholipid-PEG-ligand conjugates need to be carefully considered and optimized. 3.4.3. Density of phospholipid-PEG-ligand conjugates The density of phospholipid-PEG-ligand conjugates on the surface of nanovehicles is a vital factor that affects the targeting efficiency by altering biophysicochemical characteristics of nanovehicles and/or regulating the binding capability of ligands and receptors [115, 146]. Ding et al. synthesized DSPE-PEG2000-aptamer and prepared nanocomplexes with different contents of DSPE-PEG2000-aptamer (varying from 0, 0.5, 1, 2, 5, to 10%) [115]. Physicochemical characterization showed that the particle sizes lightly increased and ζ-potential slightly decreased with increase in the aptamer density, while encapsulation efficiency of vinorelbine remained quite constant. The uptake efficiency of nanocomplexes was significantly higher in MUC1 protein (a transmembrane glycoprotein that can specifically bind with aptamer) overexpressing cells (MCF-7) than that in MUC1 protein-negative cells (HepG2). Moreover, the targeting efficiency of nanocomplexes in MCF-7 cells was enhanced with increased aptamer density. Xu et al. prepared liposomes modified with GE11-Cys peptide (i.e., YHWYGYTPQNVIC, with specific binding to EGFR), and the resulting liposomes contained different densities of PEG (with the molar ratio ranging from 0%, 3%, 3.5%, 4%, 5%, 6%, to 8% of total lipids) and/or GE11 peptide (varying from 0%, 0.5%, 1%, to 2% of total lipids) to examine the distribution and extravasation of targeting ligand-directed liposomes in tumor tissues of SMMC-7721 xenografts [98]. Studies on the binding capability of targeting liposomes to cancer cells showed that the combination of 4% PEG and 2% GE11 peptide exhibited the best binding efficiency in SMMC-7721 cells (with EGFR overexpression), among all the formulations examined. Examination on the extravasation and cellular uptake of liposomes in tumor xenografts indicated that liposomes containing 4% PEG and 2% GE11 peptide 16

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accumulated much more efficiently inside the tumor tissues. Correspondingly, the antitumor activities of DOX-loaded liposomes with 4% PEG and 2% GE11 peptide more effectively inhibited tumor growth than other groups. Cheng et al. prepared liposomes modified with DSPE-PEG2000-GE11 peptide conjugate (with the content of GE11 peptide varied from 0%, 2.5%, 5.0%, 10.0% to 15%) for targeted drug delivery to EGFR-positive non-small cell lung cancer [147]. The physicochemical properties of these targeting liposomes, such as particle size, ζ-potential, and drug entrapment efficiency were not significantly changed compared to non-targeting liposomes. The cytotoxic effect of these liposomes in A549 tumor cells was closely related to the GE11 peptide density, and liposomes with 10% GE11 peptide displayed the highest anti-tumor activity. Generally, liposomes containing less than 10% of phospholipid-PEG-ligand conjugates in total PEGylated phospholipids can reach desirable targeting efficiency and remain their intrinsic physicochemical properties [148]. Although incorporation of targeting moieties on the surface of nanovehicles may alter their targeting efficiency in vivo, the intrinsic properties of nanovehicles (i.e., particles size, ζ-potential, morphology, and natural instincts of carriers) should be properly considered for their clinical applications.

4. Phospholipid-PEG-ligand functionalized nanomedicines for molecular imaging The development of nanotechnologies for diagnosis and/or treatment of different diseases has attracted tremendous interest in recent years and has now become an important field in multidisciplinary research. The toxicity and dose of drugs/diagnostics can be dramatically reduced by the specific targeting capability of nanomedicines to diseased sites. In vivo real-time imaging is an efficient strategy to directly observe the distribution and metabolism of nanomedicines. Near-infrared (NIR) fluorescent probe molecules (e.g., DiR, Cy5.5, Cy7.5), quantum dots (e.g., CdSe, PbS), or contrast agents (such as Fe3O4 NPs) are frequently encapsulated in nanocarriers modified with phospholipid-PEG-ligand conjugates to give rise to nanoprobes for molecular imaging of various diseases.

4.1. In vivo cancer imaging As well known, high levels of various receptors (such as FR receptor, αvβ3/αvβ5 integrin, sigma receptor, EGFR) have been detected in some tumor cells, which offer an opportunity for molecular imaging of cancers. In this aspect, nanovehicles modified with phospholipid-PEG-ligand conjugates have been extensively investigated for cancer imaging. 4.1.1. Cancer imaging using nanoprobes modified with phospholipid-PEG-small molecule ligand conjugates FA can specifically bind to FR, which is overexpressed in epidermoid carcinoma, breast cancer, hepatocellular carcinoma, cervical carcinoma, and melanoma. Nanoprobes modified with DSPE-PEG-FA have been widely employed for real-time in vivo imaging of cancers [122, 132, 139, 140, 149, 150]. For instance, DSPE-PEG-FA-decorated phytosomes containing DiR were used for in vivo imaging of H22 tumor-bearing mice [149]. In vitro cell culture studies demonstrated 17

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that the cellular uptake of DiD-MMC-loaded phytosomes was enhanced by modification with DSPE-PEG-FA conjugate. Consistently, the uptake of DiD-MMC-loaded targeting phytosomes by HeLa cells was significantly inhibited after pretreatment with excessive free FA. In vivo fluorescence imaging showed that DSPE-PEG-FA-decorated phytosomes exhibited higher fluorescence signals in tumor tissues and decreased accumulation of fluorescence in the major organs compared to those of non-targeting counterparts (Fig. 7A). Ex vivo fluorescence imaging of excised tumors and organs, microscopic observation of the cryosections of tumor tissues, in combination with quantitative analysis proved that more DiR-MMC-loaded targeting phytosomes were accumulated in tumor tissues than that of the non-targeting control (Fig. 7B-D). These results demonstrated that modification with DSPE-PEG-FA conjugate can efficiently enhance the targeting capability of phytosomes to FR-overexpressed cancer tissues. DSPE-PEG-FA-decorated NPs loaded with contrast agents are also used for MR imaging of tumors. Zhang et al. prepared DSPE-PEG-FA-coated multi-functional hybrid NPs containing Fe3O4/DTX for MR imaging in B16F10 tumor-bearing mice [151]. T2-weighted MR images showed an obvious darkening effect in tumors. Darkening of the MR images of mouse tumors was stronger than that in other organs, suggesting that the multifunctional NPs can act as desirable negative (T2) contrast agents for MR imaging. Using the post insertion method, Yang et al. synthesized Cu3BiS3 hybrid NPs modified by DSPE-PEG-FA and DSPE-PEG-chlorin e6 (Gd3+) [152]. The obtained targeting hybrid NPs exhibited effective dual-modal CT and MR imaging capacity in mice bearing HeLa tumors. At 4 h post injection of NPs, the tumor site showed obvious contrast enhancement of the CT signal from 180 ± 24 HU (before injection) to 252.3 ± 25 HU (4 h post injection), which is attributed to strong X-ray attenuation induced by Bi. The T1-weighted MR images of nude mice bearing HeLa tumors demonstrated that the MR signal intensity was enhanced by 281.6% at 6 h after injection compared to that before injection. The dual-modal CT/MR contrast functions of the hybrid NPs provides more accurate information for cancer diagnosis and location. Phospholipid-PEG-AA is another widely used PEGylated phospholipid conjugated with a small-molecule ligand, which has been employed to modify nanodiagnostics for in vivo imaging of tumors with overexpressed sigma receptors. For instance, DSPE-PEG-AA modified micelles encapsulating DiR were used for in vivo imaging of A549 tumors in nude mice [153]. The targeting DiR/micelles showed obviously stronger accumulation of DiR fluorescence signals in the tumor regions. In particular, the enhanced fluorescence signals localized in the tumor regions retained at 48 h after injection of targeting DiR/micelles. Moreover, ex vivo fluorescence imaging of excised tumor tissues further confirmed that much more accumulation of targeting DiR/micelles than non-targeting DiR/micelles in tumor sites at 48 h post injection. These results substantiated that the accumulation of nanodiagnostics in sigma receptor-overexpressed tumors was notably enhanced by decoration with DSPE-PEG-AA. In addition to the above mentioned PEGylated phospholipids with small-molecule ligands, nanoprobes decorated with 18

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DSPE-PEG-MTX and DSPE-PEG-riboflavin conjugates were examined for imaging of cervical carcinoma [59], squamous cell carcinoma [60], and prostate cancer in mice [67]. 4.1.2. Cancer imaging using NPs coated with peptide/protein-terminated phospholipid-PEG conjugates Also, nanoprobes decorated with conjugates of phospholipid-PEG and peptides or proteins are used for in vivo imaging of various tumors. Among these conjugates, DSPE-PEG-RGD conjugates have been most broadly employed to decorate nanodiagnostics for in vivo imaging of breast cancer [74, 75, 154], bone metastases [70], gastric cancer [77], and melanoma-bearing mice [155]. For example, DSPE-PEG-RGD-modified temperature-responsive liposomes containing a fluorescent probe Cy5.5 were examined for in vivo imaging of MCF-7 tumors in mice [74]. NIR fluorescence imaging demonstrated that temperature-responsive targeting Cy5.5/liposomes exhibited approximately 5 times higher accumulation in tumors than the non-targeting counterparts. This result implied that the targeting efficiency of liposomes was enhanced by decoration with DSPE-PEG-RGD conjugate, resulting from the recognition interaction between RGD and the integrin αvβ3 receptor on tumor-associated endothelial cells or tumors. In addition, DSPE-PEG-RGD conjugate modified liposomes encapsulating PbS quantum dots were explored to evaluate in vivo tumor targeting efficiency by real-time in vivo NIR fluorescence imaging [70]. NIR fluorescence images showed that the tumor accumulation of PbS/liposomes modified with DSPE-PEG-RGD conjugate was higher than that of non-targeting PbS/liposomes at various time points. Furthermore, the fluorescence intensity of tumor tissues from mice administered with targeting PbS/liposomes was 2.2-fold higher than that of the non-targeting control group at 24 h post-injection. Besides RGD peptide, tumor metastasis targeting peptide [156], lanreotide [133], luteinizing hormone-releasing hormone [78, 157], and lactoferrin are conjugated onto PEGylated phospholipids to modify nanodiagnostics for imaging of lung cancer, breast cancer, and hepatocellular carcinoma. For instance, lanreotide showed specific binding with somatostatin receptor 2. DiD-containing micelles decorated with DSPE-PEG2000-lanreotide conjugate were used for in vivo imaging of H446 tumors in mice [133]. In vivo NIR fluorescence imaging revealed that mice treated with the targeting DiD/micelles exhibited stronger fluorescence signals than those injected with non-targeting DiD/micelles (Fig. 8). Flow cytometry analyses showed that the fluorescence intensity of tumor-derived cells from mice treated with the targeting DiD/micelles was 2 times higher than that of mice injected with non-targeting DiD/micelles. These results demonstrated that the active targeting DiD/micelles can not only selectively accumulate in somatostatin receptor 2-expressing tumor tissues but also enter into tumor cells in vivo. Luteinizing hormone-releasing hormone (LHRH) receptors are overexpressed in the plasma membrane of specific cancer cells, but with extremely low expression (not detectable) in normal tissues. Micelles based on DSEP-PEG-LHRH and DSPE-PEG-Cy5.5 conjugates were used for in vivo imaging of human A549 lung carcinoma in mice [78]. The accumulation of micelles in lung tumors and major organs were detected after administration by inhalation or i.v. injection. 19

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In vivo imaging results showed that inhalation delivery enhanced accumulation of non-targeting Cy5.5/micelles in the lungs compared with that of i.v. administration. Quantitative analysis indicated that the total amount of non-targeting Cy5.5/micelles accumulated in the lungs after inhalation (83%) was more than 3.5 times higher than that subjected to i.v. injection (23%). Moreover, targeting micelles did not show significantly different distribution in other organs, as compared to non-targeting micelles. Nevertheless, targeting Cy5.5/micelles largely accumulated in the lung tumors, but non-targeting Cy5.5/micelles distributed uniformly throughout the lungs after inhalation. These results demonstrated that LHRH-mediated targeting could enhance the accumulation of micelles in the areas of lungs with tumor cells. 4.1.3. Cancer imaging using functional nanoprobes coated with phospholipid-PEG-antibody conjugates Nanodiagnostics decorated with phospholipid-PEG antibody conjugates have also been evaluated for in vivo imaging of tumors in mice. NPs functionalized with DSPE-PEG-anti-EGFR-antibody and labeled with carboxyfluorescein were used for in vivo imaging in mice bearing hepatocellular carcinoma xenografts [158]. The results indicated that targeting NPs accumulated in tumors within 1 h, and this accumulation maximized at 8 h. At 24 h, there was still a distinct accumulation for targeting carboxyfluorescein NPs in tumors, whereas little accumulation was observed for non-targeting carboxyfluorescein NPs. Furthermore, ex vivo fluorescence imaging of excised tumor tissues showed that the tumors treated with targeting NPs possessed stronger signals compared to those of non-targeting NPs. Superparamagnetic iron oxide NPs (SPIONs) decorated with DSPE-PEG-plectin-1 antibody and DSPE-PEG-Cy7 were used for dual-modality imaging of pancreatic cancer [159]. In vivo imaging showed that the T2 signal significantly decreased in pancreatic tumors at 48 h after injection with targeting SPIONs-Cy7 as compared to the pre-injection signal. By contrast, there were no significant changes in the T2 signal in tumor-bearing mice administered with non-targeting SPIONs-Cy7. Optical imaging revealed that non-targeting SPIONs-Cy7 mainly accumulated in the spleen, but targeting SPIONs-Cy7 mostly accumulated in pancreatic tumors at 48 h. These results demonstrated that fluorescence and MR dual-functional NPs modified with DSPE-PEG-plectin-1 antibody are promising for multimodality imaging of pancreatic cancers.

4.2. Molecular imaging of atherosclerotic plaques As well demonstrated, vascular cell adhesion molecule-1 (VCAM-1) is expressed by endothelial cells on the luminal surface of atherosclerotic plaques, thereby providing a rational target for diagnostic and therapeutic nanomedicines. Micelles comprised of DSPE-PEG-VCAM targeting conjugate and Cy7-labeled DSPE-PEG offered nanoprobes for in vivo imaging of atherosclerotic plaques in the early- and mid-stage in apolipoprotein E-deficient (ApoE-/-) mice [160]. In vivo studies showed that VCAM-1 targeting Cy7/micelles displayed obviously increased deposition in both early- and mid-stage aortic trees in ApoE-/- mice compared to non-targeting Cy7/micelles (Fig. 9A). Ex vivo imaging of the aortic trees of ApoE-/20

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mice treated with VCAM-1 targeting Cy7/micelles showed an approximately 2-fold increase in average fluorescence radiance, when compared with that of non-targeting Cy7/micelles in both early (p < 0.001) and mid (p < 0.01) stages of atherosclerosis, suggesting that modification with VCAM-1 targeting peptide significantly improved the accumulation of micelles in the developing plaques (Fig. 9B-C). In addition, micelles based on DSPE-PEG-VCAM-1, DSPE-PEG-Cy7, and DSPE-PEG-DTPA (Gd) were examined for simultaneous optical and MR imaging in a mouse model of atherosclerosis [161]. Clot-binding assays confirmed that micelles modified with DSPE-PEG-VCAM-1 could target clots over 8-fold higher than that of non-targeting counterpart micelles. In vivo MR and optical imaging studies on the aortas and hearts substantiated that the targeting efficiency to fibrin was significantly enhanced by the VCAM-1 targeting peptide.

4.3. In vivo imaging of spinal cord injury Spinal cord injury (SCI) is a pathological damage to the spinal cord resulting from trauma, inflammation, and other causes [162], which may lead to permanent functional sensory deficits. Due to the lack of effective therapy for SCI, targeted delivery of existing drugs or candidate agents is an important method to improve therapeutic efficacies. Apamin (a honey bee-derived peptide) exhibited high penetration and specific distribution in the central nervous system (CNS). DSPE-PEG-Apamin modified micelles encapsulating DiR were investigated for in vivo imaging of SCI [163]. After i.v. administration, DSPE-PEG-Apamin modified DiR/micelles showed significant accumulation in the site of SCI in mice, in comparison to animals treated with non-targeting DiR/micelles (Fig. 10). In line with this result, pre-injection of either free apamin or dequalinium chloride (a blocker of apamin-sensitive K+ channels) 1 h prior to administration of targeting DiR/micelles significantly reduced the distribution of DiR/micelles at the spinal cord injury site, suggesting the targeting capacity was mainly mediated by apamin via specific interaction between apamin and sensitive K+ channels.

4.4. In vivo imaging of ischemic stroke Efficient delivery of therapeutics to the injured brain region is an effective strategy to improve the cure rate of acute ischemic stroke [164]. Citicoline is a natural compound with neuroprotective effects for the treatment of neurodegenerative diseases [165]. Citicoline may also serve as a chemical exchange saturation transfer (CEST) agent [166], which can be directly detected by CEST MR imaging. Citicoline was employed by Liu et al. as a theranostic agent to directly evaluate the efficiency of drug delivery systems to ischemic regions [164]. In a rat acute ischemic injury model, citicoline-loaded liposomes (containing DSPE-PEG2000) showed elevated CEST signals in the ischemic region at 1.5 h after intra-arterial injection. Due to the abundant expression of VCAM-1 on inflamed vessels in the ischemic brain, anti-CD106 or IgG-1 antibody was conjugated onto DSPE-PEG2000-COOH decorated liposomes to target VCAM-1. As expected, 21

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immunofluorescence analysis revealed that the distribution of VCAM-1 antibody modified liposomes in the rat brain was notably higher than that of non-targeting liposomes. These results demonstrated that citicoline-containing liposomes functionalized with DSPE-PEG2000-VCAM-1 antibody can be used as a targeting and self-imaging-guided nanotherapy for the treatment of ischemic stroke. Although nanodiagnostics based on phospholipid-PEG-ligand conjugates have been broadly investigated for molecular imaging of cancer and cardio-cerebrovascular diseases in different animal models, some issues need to be addressed by in-depth studies to facilitate their clinical translation. First, the cargos of currently examined nanoprobes are mainly concentrated on NIR fluorescent molecules that are only suitable for animal experiments, but are limited for clinical applications due to their weak penetrating capacity and minor anti-interference capability in human body. Whereas a few studies have reported the applications of phospholipid-PEG-ligand decorated nanoprobes for CT, ultrasound imaging, and MR

imaging,

no

following

comprehensive

research

is

performed

in

different

animal

models.

Second,

phospholipid-PEG-ligand modified nanodiagnostics are dominantly used for imaging of various cancers and vascular diseases. Therefore, expanding their applications for precision imaging of other diseases remains an unexplored and important research field. Third, compared to the non-targeting controls, incorporation of phospholipid-PEG-ligand conjugates onto nanodiagnostics will remarkably enhance their accumulation at pathological sites, but their non-specific distribution in other major organs (such as the liver, spleen, and lung) is still unavoidable. Furthermore, reproducible and large-scale production of nanodiagnostics needs to be established by further studies.

5. Nanotherapies decorated with functional phospholipid-PEG conjugates for targeted treatment of diverse diseases The applications of nanotherapies decorated with phospholipid-PEG-ligand conjugates have attracted a great deal of interest in the past decades due to their long blood circulation characteristics and specific targeting capability. A large number of nanotherapies modified with phospholipid-PEG-ligand have been explored for targeted treatment of various cancers, orthopedic disorders, and cardiovascular diseases. The efficacies of these targeting nanotherapies have been evaluated by in vitro and in vivo experiments in different animal models. In this section, we will highlight representative nanotherapies and their in vivo applications.

5.1. Applications of nanotherapies functionalized with phospholipid-PEG-small molecule ligand conjugates Phospholipid-PEG-small molecule ligand conjugates are employed to decorate nanotherapies for targeted treatment of numerous diseases such as various cancers, staphylococcal pneumonia, and myelodysplastic syndromes (Table 1). In vitro and in vivo studies demonstrated that the efficacies of nanotherapies can be significantly enhanced by functionalization with phospholipid-PEG-small molecule ligand conjugates. 22

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5.1.1. Nanotherapies for targeted cancer treatment

Phospholipid-PEG-FA conjugate-coated nanotherapies. As well known, FRs are overexpressed in specific cancer microenvironment. Phospholipid-PEG-FA-decorated nanotherapies exhibited significantly higher accumulation and targeting efficiency in FR-overexpressed cells/tissues, compared to the corresponding non-targeting nanotherapies. Table 1 lists physicochemical properties, payloads, as well as in vitro and in vivo applications of nanotherapies decorated with phospholipid-PEG-FA conjugates. These nanotherapies are based on different nanocarriers, including liposomes, polymeric hybrid NPs, inorganic hybrid NPs, nanocomplexes, nanoemulsions, and micelles. Either anti-tumor small-molecule drugs (such as PTX, DOX, CisPt, MMC, and CPT) or nucleic acids (e.g., siRNA and DNA) can be encapsulated in these nanotherapies. Phospholipid-PEG-FA-decorated nanotherapies are usually employed to treat epidermoid carcinoma, ovarian cancer, cervical cancer, hepatocellular carcinoma, and breast cancer, due to overexpressed FRs on these cancer cells. The internalization and accumulation of phospholipid-PEG-FA-decorated nanotherapies have been evaluated in FR-overexpressed cell lines such as human epidermal carcinoma (KB), human cervical carcinoma (HeLa), human breast carcinoma (MCF-7), and human ovarian carcinoma (SKOV-3). Observation by confocal laser scanning microscopy (CLSM) proved that the internalization and accumulation of targeting nanotherapies were considerably enhanced in FR-overexpressed cells through FA-mediated endocytosis. In vitro cell culture experiments demonstrated that phospholipid-PEG-FA-modified nanotherapies exhibited increased anti-tumor activities compared to non-targeting nanotherapies. Futhermore, in vivo efficacy and safety of phospholipid-PEG-FA-modified nanotherapies were evaluated using mouse models of tumor xenografts of FA overexpressed cancer cells, including KB, MCF-7, HeLa, H22, B16F10, S180, and drug resistant KBv carcinoma cells. The targeting nanotherapies showed a significantly stronger anti-tumor growth effect than non-targeting nanotherapies and free drug. Moreover, phospholipid-PEG-FA-decorated nanotherapies exhibited excellent biocompatibility. For example, Hou el al. developed DSPE-PEG-FA decorated MMC/liposomes for targeted therapy of hepatocellular carcinoma in mice [149]. It was found that the targeting MMC/liposomes showed an intensive tumor growth inhibition rate compared to the non-targeting MMC/liposomes and free drug. Moreover, the targeting liposomes displayed lower side effects than free MMC after i.v. administration. Mice treated with free MMC exhibited obvious body weight loss, and animal death was observed. By contrast, all the animals treated with the targeting MMC/liposomes survived throughout the experiments. These results demonstrated that the adverse effects of chemotherapeutics can be efficiently suppressed by formulating into targeting liposomes with DSPE-PEG-FA decoration. Consequently, the DSPE-PEG-FA-modified nanoplatform is an effective drug delivery system with great therapeutic potential. Due to its structural similarity with FA, MTX is also used as a targeting ligand to the FA receptor. Nanotherapies decorated with phospholipid-PEG-MTX conjugate were investigated to treat FR overexpressed cancers [59, 60]. In vivo 23

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studies proved that phospholipid-PEG-MTX-modified nanotherapies exhibited synergistic anti-tumor effects and reduced side effects, thereby affording the improved therapeutic index. Phospholipid-PEG-AA conjugate-coated nanotherapies. Phospholipid-PEG-AA-decorated nanotherapies are widely studied to treat sigma receptor-overexpressed tumors, such as melanoma, prostate cancer, and lung cancer. Physicochemical characteristics, morphology, and payloads of nanotherapies modified with phospholipid-PEG-AA conjugates and their in vitro/in vivo applications are summarized in Table 1. Tumor cells with high expression of sigma receptors, such as DU-145, H460, and B16F10 cells were selected to evaluate the cellular internalization of phospholipid-PEG-AA-modified nanotherapies. Compared to non-targeting counterparts, nanotherapies decorated with phospholipid-PEG-AA exhibited more efficient cellular uptake by sigma receptor-overexpressed cells. Correspondingly, there was no significant difference in cellular uptake in HUVECs (with low sigma receptor expression) between targeting and non-targeting nanotherapies [57]. These results substantiated that the enhanced cellular uptake of phospholipid-PEG-AA modified nanotherapies was partially mediated by the sigma receptors. In vivo anti-tumor efficacy of phospholipid-PEG-AA-modified nanotherapies was assessed in mice bearing sigma receptor-overexpressed tumors. Incorporation of phospholipid-PEG-AA onto nanotherapies obviously strengthened their anti-tumor activity compared to non-targeting nanotherapies and free drugs. For instance, DSPE-PEG-AA-decorated NPs loaded with cytochrome C were examined to treat H460 tumors in mice [167]. Treatment with targeting NPs apparently inhibited tumor growth, while therapy with free drug resulted in partial tumor growth retardation. Polymeric metformin and cisplatin (CisPt)-loaded NPs were also studied in mice bearing H460 xenografts [168]. In this case, DSPE-PEG-AA-decorated nanotherapies exhibited significantly enhanced anti-tumor efficacy and reduced systemic toxicities in comparison to free drug and non-targeting nanotherapies. NPs decorated with phospholipid-PEG-AA could remarkably enhance targeting efficiency for sigma receptor-overexpressed cancer cells and provide protection for unstable drugs. 5.1.2. Nanotherapies for antibacterial applications Since some antibacterial agents can identify the outer structure of the pathogen-like cell wall and/or cell membrane, DSPE-PEG conjugated with antibacterial agents have been utilized for targeted delivery of antibiotics to certain bacteria [64]. Li et al. employed DSPE-PEG-daptomycin-modified liposomes encapsulating daptomycin for treatment of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Flow cytometry analysis indicated that liposomes modified with DSPE-PEG-daptomycin conjugate showed specific binding to MRSA. In vivo whole-body fluorescence imaging in mice demonstrated that DSPE-PEG-daptomycin modified liposomes exhibited good targeting capability in vivo to MRSA-infected lungs in a pneumonia model. The targeting liposomes loaded with daptomycin not only prolonged the survival time of mice, but also exhibited more favorable antibacterial efficacy against MRSA than conventional PEGylated 24

liposomal daptomycin.

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Activated macrophages play acrucial role in the defense against invasive bacteria [169]. FA exhibits strong affinity to FRs which are also overexpressed on activated macrophages [170]. Consequently, DSPE-PEG3500-FA-coated NPs encapsulating antibiotics were employed to target macrophages for drug delivery to the infected site [171]. In vitro cellular assay verified that moxifloxacin-loaded NPs with DSPE-PEG3500-FA coating significantly eradicated resident bacteria in macrophages compared to their non-targeting counterpart. In a mouse model of pulmonary P. aeruginosa infection, DSPE-PEG-FA-modified nanotherapies showed better antibacterial efficacy than free drug and non-targeting nanotherapies. Importantly, the survival time of infected mice was prolonged by treatment with DSPE-PEG-FA-modified nanotherapeutics. 5.1.3. Nanotherapies for treatment of bone marrow diseases Alendronate (ALN), a bisphosphonate, exhibits strong chelation with calcium ion of mineral hydroxyapatite that is the main ingredient of bone marrow niches. Therefore, alendronate has been frequently selected as a targeting moiety for targeted treatment of bone marrow diseases. NPs modified with DSPE-PEG-ALN were examined for co-delivery of decitabine (DAC) and arsenic trioxide (ATO) to treat myelodysplastic syndrome (MDS) (Fig. 11A) [63]. In vivo imaging in a mouse model of MDS showed that bone-targeting NPs (BTNPs) mainly accumulated in all bone tissues (femur, spine, and skull) compared to those treated with non-targeting NPs at 2 h after i.v. injection (Fig. 11B). Biodistribution analysis proved that modification with DSPE-PEG-ALN facilitated accumulation of DAC and ATO in the bone, which is 6.7 and 7.9 times more than that of non-targeting NPs (Fig. 11C). Four weeks after BTNPs treatment of MDS in mice, counts of white blood cells (WBCs), red blood cells (RBCs), and platelets (PLTs) significantly increased compared to those of mice treated with non-targeting NPs or free drugs (Fig. 11Diii-v). Furthermore, mouse survival was significantly prolonged by treatment with BTNPs, compared to that of mice treated with non-targeting NPs or free drugs (Fig. 11Dvi). Consistently, quantitative PCR analysis revealed that the expression of DNA methyltransferase 1 (DNMT1) mRNA in bone marrow cells was evidently decreased after treatment with BTNPs. Also, treatment with BTNPs resulted in the lowest level of γH2AX (a biomarker for DNA damage) in MDS bone marrow cells (collected from MDS mice). In addition, significantly higher apoptosis of bone marrow cells was detected after treatment with BTNPs, in comparison to those treated with free drug or non-targeting NPs. These results demonstrated that surface modification with DSPE-PEG-ALN can remarkably increase the targeting capability and therapeutic efficacy of NPs for treatment of bone marrow diseases.

5.2. Applications of nanotherapies functionalized with phospholipid-PEG-peptide conjugates As targeting moieties, peptides have numerous advantages such as stable structure, easy modification, and small size, which are favorable for modification of nanotherapies [18]. Due to specific binding affinity of peptides and their receptors, 25

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nanotherapies decorated with phospholipid-PEG-peptide conjugates are able to specifically accumulate in atherosclerotic plaques [160, 161], spinal cord injury sites [163], tumors [41, 87], and brain[81]. In vivo studies demonstrated that targeting nanotherapies exhibit low toxicity and potentiated efficacies compared to non-targeting nanotherapies or free drugs. 5.2.1. Targeted cancer therapy To functionalize nanotherapies, PEGylated phospholipids are conjugated with various peptides, such as cell penetrating peptide, transferrin receptor targeting peptide, tumor metastasis targeting peptide, tumor-penetrating peptide, EGFR targeting peptide, vasoactive intestinal peptide, and angiogenic vessel targeting peptide. Representative in vitro and in vivo applications of nanotherapies decorated with the phospholipid-PEG-peptide conjugates are summarized in Table 2. In vivo efficacies of these nanotherapies were evaluated in different models of tumors, including breast cancer, glioma, liver cancer, lung cancer, prostate cancer, colon cancer, and acute myeloid leukemia. Nanotherapies functionalized with phospholipid-PEG-RGD conjugates. RGD can selectively bind to αvβ3/αvβ5 integrins, which

are

upregulated

on

neovasculature

endothelial

cells.

Therefore,

nanotherapies

decorated

with

phospholipid-PEG-RGD conjugates have been broadly examined for targeting of tumor-associated neovasculature. The internalization and accumulation of nanotherapies coated with phospholipid-PEG-RGD conjugates were assessed using integrin upregulated cell lines, such as mouse prostate cancer cells (RM-1), human retinal pigment epithelial cells (ARPE-19), human hepatoma cells (HepG2), mouse melanoma cells (B16F10), human breast cancer cells (MDA-MB-435s), human primary glioblastoma cells (U87MG), mouse breast tumor cells (4T1), and HeLa. To observe cellular internalization of phospholipid-PEG-RGD modified NPs, Liu et al. chosen MDA-MB-435s cells (integrin αvβ3 positive) and MCF-7 cells (integrin αvβ3 negative) for cell culture studies [172]. MDA-MB-435s cells incubated with DSPE-PEG-RGD modified hybrid NPs containing coumarin exhibited higher cellular fluorescence intensities than those incubated with non-targeting coumarin-loaded hybrid NPs. The mean fluorescence intensity of targeting NPs was approximately 2.7-fold higher than that of the non-targeting control NPs. By contrast, there was no significant difference in the cellular fluorescence intensity in integrin αvβ3 negative MCF-7 cells after separate treatment with either targeting or non-targeting NPs. In line with these results, anti-tumor cytotoxicity of phospholipid-PEG-RGD decorated nanotherapies was remarkably strengthened in integrin upregulated cells via RGD-mediated targeting. Mice bearing various tumors (such as melanoma, bone metastases, and breast cancer) have been chosen to evaluate in vivo anti-tumor efficacies of phospholipid-PEG-RGD modified nanotherapeutics. Enhanced anti-tumor efficacy was achieved by targeting nanotherapies compared to non-targeting nanotherapies and free drugs. For example, DSPE-PEG-RGD-modified NPs encapsulating both hexadecyl-oxaliplatin-trimethyleneamine and a chlorin e6 derivative were used for treatment of orthotopic 4T1 tumor-bearing mice (Fig. 12Ai) [154]. It was found that the targeting nanotherapy more effectively reduced tumor growth as compared to the non-targeting control (Fig. 12Aii). 26

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Nanotherapies based on phospholipid-PEG conjugated with other peptides. In addition to RGD, other tumor-targeting peptides are conjugated onto PEGylated phospholipids to modify nanotherapies for targeted cancer treatment. For example, α-conotoxin ImI shows high affinity to Alpha7 nicotinic acetylcholine receptor (a7 nAChR) that is specifically expressed in cancers. Based on the specific targeting capability of α-conotoxin ImI, PTX-loaded NPs modified with DSPE-PEG-α-conotoxin ImI conjugate were used to treat MCF-7 tumors in mice (Fig. 12B) [173]. The growth of MCF-7 tumors was markedly inhibited by PTX-loaded targeting NPs compared to the non-targeting counterpart. Moreover, the PTX-loaded targeting NPs showed notably low systemic toxicity compared to free drug. DOX-loaded liposomes modified with a conjugate of DSPE-PEG and tumor metastasis targeting peptide also significantly suppressed tumor growth and exhibited minor side effects in treatment of MDA-MB-231 tumors in mice (Fig. 12C) [156]. Interestingly, adoptive transfer of cytotoxic T lymphocytes (CTLs) has been used as an immunotherapy in melanoma, but the specificity for tumors is undesirable. Interleukin-4 receptor (IL-4R) is up-regulated in melanoma. Therefore, CTLs coated with a conjugate of DOPE-PEG and IL-4R targeting peptide were used for treatment of melanoma-bearing mice (Fig. 12D) [174]. In vivo experiments demonstrated that IL-4R-targeting CTLs efficiently inhibited melanoma growth and prolonged the survival rate of mice. These results substantiated that modification of nanotherapies with phospholipid-PEG-peptide conjugates is a promising strategy to improve their anti-tumor activity and decrease side effects. 5.2.2. Treatment of ocular diseases Based on specific binding to retinal cells, DSPE-PEG-RGD modified micelles encapsulating flurbiprofen (FB) were used for treatment of ocular diseases (Fig. 13A) [175]. These micelles showed rapid mucoadhesion on the ocular surface through interactions between cRGD and the corneal epithelium, with long ocular surface retention time and robust transcorneal penetration ability (Fig. 13B). FB-loaded targeting micelles more effectively inhibited ocular inflammation progression compared to commercial flurbiprofen eyedrop (0.03% flurbiprofen sodium, FBNa) and non-targeting FB micelles at the same dose, suggesting that a reduced dose cana maintain effective efficacy (Fig. 13C-D). Ocular safety evaluation revealed no significant difference in the central corneal thickness between the targeting FB micelles, non-targeting FB micelles, and free FB (Fig. 13E). H&E staining of retinal sections indicated that all the retinal architectures were well organized after treatment with free drug or FB-loaded micelles. Eyes treated with FB-loaded micelles showed no apparent defects or inflammatory exudates in the outer nuclear layer, inner nuclear layer, and the retinal pigment epithelium (RPE) of the retina. These results affirmed that DSPE-PEG-RGD modification can improve the targeting efficiency of micelles to retinal cells and improve their anti-inflammatory activity in ocular diseases. 5.2.3. Treatment of thrombosis Also, DSPE-PEG-RGD functionalized liposomes loaded with urokinase were used to treat thrombus [176]. The binding ability of DSPE-PEG-RGD-modified liposomes to activated platelets was affirmed by flow cytometry. Platelets can be 27

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activated by thrombin, and high expression of P-selectin was detected on platelet membranes. In vitro experiments indicated that liposomes modified with DSPE-PEG-RGD exhibited special affinity to activated platelets. The thrombolytic efficacy of DSPE-PEG-RGD-modified urokinase/liposomes was evaluated in a rat model of thrombosis. In vivo thrombolysis results demonstrated that DSPE-PEG-RGD-modified liposomes at a reduced dose of urokinase by 75% can achieve an equivalent thrombolytic effect as compared to free urokinase in a mouse model of mesenteric thrombosis. 5.2.4. Treatment of vascular inflammatory diseases Antimicrobial peptides (AMPs) are found from bacterial to mammalian species, which have been examined to inhibit the destructive effects of proinflammatory cascades. Due to their severe side effects, such as hemolysis, nephrotoxicity, and neurotoxicity, AMPs cannot be directly used as safe drugs for treating inflammatory diseases. Recently, a low hemolytic and broad-range antibiotic AMP (i.e., KSLW peptide, KKVVFWVKFK) was selected and conjugated onto DSPE-PEG to form micelles for treating sepsis (Fig. 14A) [177], a disease closely related to inflammatory responses in vascular endothelium resulting from bacterial infection. Micelles based on DSPE-PEG-KSLW conjugate not only improved the survival rate of mice with induced sepsis, but also inhibited lipopolysaccharide (LPS)-induced severe vascular inflammatory responses in human umbilical vein endothelial cells, with significantly potent efficacies compared to free KSLW or PEG-KSLW conjugate (Fig. 14B). Furthermore, DSPE-PEG-KSLW-derived micelles dramatically reduced the bacterial count and inhibited bacterial growth (Fig. 14C-F). These results suggested that DSPE-PEG-KSLW-derived micelles can be further developed to treat severe vascular inflammatory diseases, such as sepsis and septic shock. Abdominal aortic aneurysm (AAA), a chronic inflammatory disease, is a leading cause of mortality and morbidity in the elderly, affecting about 5% of the population in developed countries [178]. Pathologically, infiltration of inflammatory cells, elevated expression of matrix metalloproteinases (MMPs), over-production of reactive oxygen species (ROS), intimal calcification, neovascularization, and apoptosis of vascular smooth muscle cells (VSMCs) are frequently involved in aneurysms [179, 180]. Using a conjugate of DSPE-PEG and a cyclic peptide ligand cRGDfk, our group fabricated ROS-responsive, rapamycin-loaded NPs based on a hydrophobic cyclodectrin material for targeted treatment of AAA (Fig. 15A) [181]. In vitro cellular internalization assay demonstrated that DSPE-PEG-cRGDfK functionalized, Cy5-labeled fluorescent NPs exhibited significantly higher cellular uptake as compared to the undecorated control in VSMCs (Fig. 15B). In vivo targeting efficiency of the DSPE-PEG-cRGDfK-functionalized ROS-responsive NPs was assessed in rats with CaCl2-induced AAA. Ex vivo imaging of aneurysmal sites showed that the fluorescent intensity of DSPE-PEG-cRGDfK conjugate-coated NPs was 2.5-fold higher than that of cRGDfK-deficient NPs at 8 h post i.v. injection (Fig. 15C). Notably, DSPE-PEG-cRGDfK-coated NPs displayed relatively high accumulation in both intimal and medial regions of abdominal aortas. These results substantiated that the aneurysmal targeting capability of NPs can be considerably enhanced by decoration

with

DSPE-PEG-cRGDfK.

Further

in

vivo 28

efficacies

in

AAA

rats

demonstrated

that

the

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DSPE-PEG-cRGDfK-coated rapamycin nanotherapy displayed significantly higher anti-aneurysmal activity than that of the non-targeting counterpart (Fig. 15D), in terms of attenuating the expansion of aortic diameter, preventing calcification, decreasing elastin degradation, and maintaining endothelial integrity, as well as dampening inflammation and lowering oxidative stress in aneurysmal tissues. 5.2.5. Treatment of spinal cord injury As previously mentioned, apamin exhibits high penetration and specific accumulation in the central nervous system (CNS). Therefore, curcumin-loaded micelles modified with a DSPE-PEG-apamin conjugate were used for treatment of spinal cord injury [163]. In vivo pharmacodynamic studies showed that apamin-mediated targeting nanotherapies not only enhanced recovery but also prolonged survival of mice with spinal cord injury, as compared to the non-targeting or control groups. Moreover, targeting micelles can decrease the drug toxicity, indicating that DSPE-PEG-apamin-modified nanotherapies have great potential for treatment of central nervous system diseases. 5.2.6. Treatment of Parkinson’s disease Chlorotoxin, a 36-amino acid peptide, can specifically bind to the brain gliomas and proliferating vascular endothelial cells. DSPE-PEG-chlorotoxin conjugate was employed to decorate liposomes encapsulating levodopa for targeted therapy of Parkinson’s disease [83]. In vitro cellular assay demonstrated that modification with DSPE-PEG-chlorotoxin notably facilitated the uptake of liposomes by brain microvascular endothelial cells. Biodistribution experiments proved that targeting liposomes loaded with levodopa significantly increased the distribution of the metabolites of levodopa (such as dopamine and dihydroxyphenyl acetic acid) in the substantia nigra and striata. In a mouse model of Parkinson’s disease induced

with

methyl-phenyl-tetrahydropyridine

(MPTP),

DSPE-PEG-chlorotoxin-modified

levodopa/liposomes

significantly attenuated the serious behavioral disorders and diminished the MPTP-induced loss of tyrosine hydroxylase-positive dopaminergic neurons. These results demonstrated that modification with DSPE-PEG-chlorotoxin is able to improve the brain targeting capability of NPs for therapy of Parkinson’s disease. 5.2.7. Prevention of osteoporosis Asp8, an oligopeptide of eight aspartate residues showing specific targeting to the bone, was conjugated onto DSPE-PEG to prepare bone-targeting liposomes encapsulating icaritin for treatment of osteoporosis [182]. In vivo imaging showed that the fluorescence signal in the femurs strongly increased after injection of DiR-labeled bone-targeting liposomes compared to the non-targeting counterparts in the first 24 h. After 72 h of injection, the targeting liposomes remained stronger fluorescence signals in the spine and femur than those of the non-targeting control. Observation by ex vivo micro-CT showed that ovariectomized mice treated with bone-targeting icritin-loaded liposomes had significantly higher bone mineral density, bone volume ratio, and connectivity density than those of the non-targeting group. Furthermore, the targeting icaritin nanotherapy can enhance the bone formation in ovariectomized mice compared to the control icaritin 29

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nanaotherapy without the Asp8 moiety. These results demonstrated that Asp8-modified liposomes can function as a bone-targeting delivery system to effectively deliver osteogenic therapeutics and enhance their efficacies for prevention of estrogen depletion-induced osteoporosis.

5.3. Nanomedicines functionalized with phospholipid-PEG protein conjugates Proteins, such as lactoferrin, transferrin, ephrin-A1, and fibronectin extra domain B are used as targeting moieties due to their receptors are highly expressed in specific tumor cells, but expressed at low levels in most normal cells. Therefore, phospholipid-PEG-protein conjugates have been employed to functionalize nanotherapies to increase their targeting capability. Typical applications of nanotherapies modified with phospholipid-PEG-protein conjugates are summarized in Table 3. These nanotherapies are mainly used for molecular imaging and targeted therapy of various cancers. In vitro antitumor activities of these targeting nanotherapies are generally assessed in transferrin receptor-overexpressed cancer cells (e.g., HCT-8, Colon 26, HepG2, A549, and K562 cells), ephrin type-A receptor 2-overexpressed cancer cells (such as NSCLC cells), and fibronectin extra domain B-overexpressed cancer cells (e.g., U87MG, SCC-7, GL26, NIH3T3, LLC, and LNCaP cells). In vivo antitumor efficacies of nanotherapies modified with DSPE-PEG-protein were evaluated in hepatocellular carcinoma [86], lung cancer [183], and glioma [102] with overexpressed specific protein receptors. For example, fibronectin extra domain B (EDB) is an extracellular matrix biomarker, which is also recognized as a novel tumor-associated biomarker due to its specific expression in tumor-associated blood vessels and even on cancer cells [184, 185]. An aptide specific to EDB (defined as APTEDB) was conjugated to DSPE-PEG-Mal to fabricate EDB-targeting liposomes [102]. In vivo antitumor efficacy of DOX-loaded liposomes modified with DSPE-PEG-ATPEDB with different PEG pairs was assessed in a U87MG xenograft model. DSPE-PEG2000-APTEDB/PEG1000 modified DOX/liposomes showed the strongest antitumor activity, which retarded tumor growth to the greatest extent (~90%), followed by DSPE-PEG1000-APTEDB/PEG550 modified DOX/liposomes (~80%). Also, both DSPE-PEG2000-APTEDB/PEG2000 and DSPE-PEG1000-APTEDB/PEG1000 modified DOX/liposomes significantly

suppressed

tumor

growth

compared

to

the

non-targeting

DOX/liposomes.

In

addition,

DSPE-PEG-APTEDB-modified DOX/liposomes were examined to treat GL26 tumors in mice [103]. Compared to free drug and the non-targeting DOX/liposomes, treatment with DSPE-PEG-APTEDB-modified DOX/liposomes resulted in significantly smaller tumor volume. Likewise, the tissue distribution and therapeutic effects of DOX-loaded liposomes coated with DSPE-PEG-transferrin conjugate were investigated in liver tumor-bearing mice [86]. At all the time points examined, remarkably increased DOX levels were detected in tumor tissues from mice treated with the targeting DOX/liposomes, in comparison to those treated with the non-targeting DOX/liposomes and free DOX. In vivo tumor growth was apparently suppressed by DOX/liposomes 30

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modified with DSPE-PEG-transferrin conjugate. The average weight of excised tumors was approximately 330 and 1170 mg for mice treated with the targeting and non-targeting liposomes, respectively. In addition to antitumor applications, nanotherapies decorated with DSPE-PEG-protein were explored for antithrombotic therapy. For example, since thrombomodulin can act as a major cofactor in the protein C anticoagulant pathway, DSPE-PEG-thrombomodulin conjugate-functionalized liposomes were examined for antithrombotic therapy [43]. The targeting nanotherapy showed considerable in vivo anticoagulant effects by decreasing the mortality from 80% to 20% in a mouse model of thromboembolism induced by thrombin. Accordingly, this targeting liposomal formulation with long blood circulation can serve as a potential effective anticoagulant agent.

5.4. Nanotherapies based on phospholipid-PEG antibody conjugates 5.4.1. Targeted cancer treatment Antibodies have been extensively used in the development of targeting nanotherapies, due to their high specificity and affinity to the corresponding targets. The antigen-binding fragment (Fab), single-chain antibody fragment (ScFv), and monoclonal antibody (mAb) are frequently conjugated onto PEGylated phospholipids to decorate nanotherapies. Cancer cells (such as KG1, MCF-7, B16, SMMC-7721, HepG2, Huh7, BxPC-3, and XPA-3 cell lines) with specific antigen overexpression were selected to evaluate cellular internalization and in vitro anti-cancer activities of nanotherapies modified with phospholipid-PEG-antibody conjugates. For example, in vitro antitumor effects of ADM-loaded NPs modified with DSPE-PEG-anti-VEGFR-2 antibody were investigated in hepatic cellular cancer cells [158]. In vitro activity of ADM/NPs was notably enhanced by modification with DSPE-PEG-anti-EGFR-antibody. The IC50 value of targeting ADM/NPs was 2-fold lower than that of non-targetingg ADM/NPs in SMMC-7721 and HepG2 cancer cells. In addition, in vivo antitumor efficacies of nanotherapies modified with phospholipid-PEG-antibody were evaluated in animal models. The targeting nanotherapies generally exhibited enhanced antitumor activities and prolonged survival time compared to the non-targeting controls. Guo et al. employed ADM-loaded hybrid NPs decorated with DSPE-PEG-anti-EGFR antibody for therapy of hepatocellular carcinoma [158]. In vivo studies demonstrated that the targeting ADM nanotherapy more significantly suppressed tumor growth than the non-targeting ADM nanotherapy or free drug. The mean tumor size was 116 ± 75, 479 ± 118, and 625 ± 153 mm3 for mice treated with the targeting nanotherapy, non-targeting nanotherapy, and free ADM, respectively. 5.4.2. Targeted treatment of atherosclerosis Lectin-like oxidized low-density lipoprotein receptor-1 (LOX1) is a potent regulator of atherosclerosis, and therefore it is developed as a new target for targeted therapy of this vascular disease. In this aspect, fasudil-loaded liposomes modified with DSPE-PEG-anti LOX1 antibody were used for targeted treatment carotid artery lesions [186]. The results showed that 31

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targeting fasudil/liposomes notably inhibited the inflammatory reaction, such as smooth muscle cell infiltration and foam cell formation in carotid hypertrophy. As compared to the non-targeting control, LOX1-targeting liposomal fasudil more significantly reduced the intimal thickness. In conclusion, compared with non-targeting nanomedicines, nanotherapies decorated with functional phospholipid-PEG conjugates exhibited desirable therapeutic effects in animal models of various diseases, largely resulting from prolonged circulation time and specific targeting capability of nanovehicles. Nevertheless, the effectiveness of these nanomedicines was mainly evaluated in murine models, which are significantly different from the real disease situations in patients. Moreover, many issues such as long-term toxicity and integrated pharmacokinetic/pharmacodynamic profiles remain to be elucidated by comprehensive studies for their clinical translation.

6. Conclusions and future prospects Over the past decades, various phospholipid-PEG-derived functional conjugates have been synthesized, owning to their multiple advantages such as good biocompatibility, long blood circulation character, and easy modification of different nanovehicles. Decoration of nanodiagnostics and nanotherapies with functional phospholipid-PEG conjugates can not only prolong their circulation time in the blood, but also improve targeting efficiency to diseased sites. Different factors, such as the length and pairs of PEG, types of PEGylated phospholipid linkers, fabrication methods, and surface density of functional phospholipid-PEG conjugates can affect biophysicochemical properties and targeting efficiency of the resulting nanodiagnostics and nanotherapies. These targeting nanomedicines have been extensively investigated and developed for molecular imaging and targeted therapy of a diverse array of diseases. Nevertheless, there is still a long way to go for the clinical translation of the majority of these targeting nanoprobes and nanotherapies. For example, the biophysicochemical properties as well as reproducible and scalable preparation methodologies of nanodiagnostics and nanotherapies decorated with functional phospholipid-PEG conjugates need further intensive investigation. In addition to 2D culture-based in vitro cellular evaluations, 3D culture and organoid models can be used for in vitro studies, in order to provide more useful data. For the animal models used in numerous in vivo studies, they cannot perfectly reflect the real conditions in patients due to their multiple liminations, frequently leading to false positive results that cannot be repeated in clinical trials. To get more reliable data for further translation studies, the safety, biodistribution, and pharmacokinetic/pharmacodynamic profiles of these targeting nanodiagnostics and nanotherapies must be comprehensively investigated in multiple animal species based on rationally designed experimental regimens and using well-recognized controls.

Acknowledgements 32

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This work is financially supported by the National Natural Science Foundation of China (Nos. 31101447 & 81971727).

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45

Journal Pre-proof Table 1. Nanodiagnostics and nanotherapies decorated with phospholipid-PEG-small molecule ligand conjugates. Phospholipid-PEG -ligand conjugates

DSPE-PEG-FA

DPPE-PEG-FA

DSPE-PEG-AA

DSPE-PEG-cholic acid DSPE-PEG-MTX DSPE-PE-sialyl Lewis X analogs DSPE-PEG-ribofla vin derivatives DSPE-PEG-butyra te DSPE-PEG-dapto mycin DSPE-PEG- ALN DSPE-PEG-oestro ne DSPE-PEG-dopa mine DSPE-PEG-borne ol DSPE-PEG-mann ose DSPE-PEG-dequal inium

Nanovehicles

Size (nm)

Payloads

Cells/tissues

In vivo applications

References

Liposomes

33-626

PTX, DOX, DTX, DNA, oxaprozin, MMC, CPT, curcumin, ODNs, vincristine, gadolinium, etoposide, Fe3O4, Imatinib, and 5-Fu

RAW264.7, KB, KBv, KB31, KB85, HT-1080, HeLa, MCF-7, A549, HEK293, Caco-2, C6, MV4-11, IGROV-1, T24, J6456, M109R, C26, MCF-7, M109, A375, NHF, and PC-3 cells

Oral cancer, hepatocellular carcinoma, aascites tumor, lymphoma, melanoma, breast cancer, colon cancer, cervical carcinoma

[48-51, 53-56, 149, 151, 187-205]

Micelles

12-230

DOX, DTX, CPT, and tripentone

Oral cancer

[131, 132, 206-208]

Polymer hybrid NPs

65-280

siRNA, DTX, DOX, PTX, 90Y, ICG, CisPt, and MXF

Liver tumor, oral cancer, ovarian cancer, breast cancer, pulmonary infection

[122-127, 150, 171, 209-212]

Inorganic hybrid NPs

73-200

Nanocomplexes

19-200

Nanoemulsions

105-130

Nano suspensions

~120

Nanosheets

~4

Carbon nanotubes Nanocomplexes

~39

Liposomes

114-117

Gadolinium, CdSe, Ag2S, and Cu3BiS3 Bismuth, MMC, curcumin, and siRNA All-trans retinoic acid and aclacinomycin A Annonaceous acetogenins Black phosphorus nanosheet Alexa647 DNA DOX and siRNA

KB, KBv, HepG2, SKOV-3, HeLa, SGC7901, and BXPC3 cells KB, MCF-7, MDA-MB-231, KB, A549, OVCAR-3, SW626, SKOV-3, COS-7, EMT6, LNCaP, VCaP, NIH, and 3T3 cells KB, KB-FR, C26, J6456-FR, HeLa, A549, and MCF-7 cells KB, 4T1, HDF, Hela, Caco-2, 3T3, and LNCaP cells

Micelles

20-63

Polymeric hybrid NPs

189-279

Nanocomplexes

20-235

Exosomes

76-304

Liposomes

90-100

DOX and silybin

[128, 129, 152, 213] [138-143]

KB, MCF-7, and HepG2 cells

Oral cancer

[134, 214, 215]

A549 cells

Cervical cancer

[216]

HeLa cells

Cervical cancer

[217]

T24 cells BNL CL2, CHO, and HeLa cells

Prostate cancer and melanoma

[136] [52]

Du-145 and B16F10 cells

Cytochrome C and siRNA P53, CisPt, and metformin CisPt, acyclovir, and ursolic acid PTX

Oral cancer, peritoneal tumor, cervical cancer Breast cancer and cervical cancer

[58, 218]

H460 and H1299 cells

Lung cancer

[167, 219]

A549, BEAS-2B, and H460 cells

Lung cancer

[153, 168]

NCI-H446, A549, H460, HUVECs, and B16F10 cells 3LL-M27 cells

Lung cancer and melanoma Lewis lung carcinoma

[57, 220, 221] [222]

Health rats

[65, 66]

Caco-2 and HepG2 cells

Micelles

20-40

SAL, MMC, and curcumin

HeLa, A549, CAL-27, and FaDu cells

Nanocomplexes

~173

Zinc phthalocyanine

HeLa and MCF-7 cells

Cervical carcinoma and squamous cell carcinoma Breast cancer

Liposomes

96-106

Fluorescein

HUVECs

-

[61]

Liposomes

~141

DiR and Rhodamine B

HUVEC, PC3, and A431 cells

Prostate cancer

[67]

Hybrid NPs

80–90

Insulin

E12 and Caco-2 cells

Health rats

[225]

Staphylococcal pneumonia Myelodysplastic syndromes

[59, 60, 223] [224]

Liposomes

86-94

Daptomycin

Methicillin-resistant S. aureus

[64]

Nanocomplexes

80-120

Decitabine and arsenic trioxide

Bone marrow cells

Liposomes

~140

MTX

HL-60 cells

-

[226]

Liposomes

~160

Mangiferin

HUVECs cells

Skinflap regeneration

[227]

[63]

Nanomicelles

~15

DOX

HBMEC cells

Glioblastoma

[228]

Liposomes

~173

[30]

~220

RAW264.7 cells Dendritic cells and cytotoxic T lymphocytes

-

Hybrid NPs

Ovalbumin Monophosphoryl lipid A and imiquimod

E.G7-OVA tumor

[229]

Liposomes

~120

Resveratrol

B16F10 cells

-

[230]

46

Journal Pre-proof

Table 2. Different nanomedicines decorated with phospholipid-PEG-peptide conjugates. Phospholipid-PEGpeptide conjugates

Nanovehicles

Liposomes

Size (nm)

84-259

Payloads

Cells/tissues

In vivo applications

CisPt, PbS, vincristine, DOX, 5-FU, L-cysteine, gemcitabine, combretastatin A4, matrine, RNA, DNA,

RM-1, U87MG, HepG2, HUVEC, SGC7901, BGC823, MGC803, GES1, RPECs, B16, B16F10, HUVEC, MDA-MB-231, Huh7, BxPC-3, A375, HT-29, Bcap-37, EPCs, and platelets

Prostate cancer, breast cancer, glioma, hepatocellular carcinoma, melanoma, gastric cancer, and mesenteric vessel thrombosis

MCF-7 cells

-

HCECs A549, MCF-7, MDA-MB-435s, VSMCs, and RAW264.7 cells

Ocular inflammation Lung cancer and abdominal aortic aneurysms Breast cancer and lung cancer

and sunitinib DSPE-PEG-RGD

DSPE-PEG-Luteini zing hormone-releasing hormone DSPE-PEG-t transferrin receptor targeting peptide

DSPE-PEG-cell penetrating peptide

DOPE-PEG-cell penetrating peptide DSPE-PEG-Transc riptional transactivator peptide DSPE-PEG-tumor metastasis targeting peptide DSPE-PEG-EGFR targeting peptide DSPE-PEG-a-cono toxin ImI DSPE-PEG-chlorot oxin DSPE-PEG-vasoac tive intestinal peptide DSPE-PEG-bombe sin peptide analogue DSPE-PEG-bradyk inin DSPE-PEG-antago nist G DSPE-PEG-octreot ide DSPE-PEG-bombe sin DSPE-PEG-lanreot ide DSPE-PEG-apami n DSPE-PEG-angiop ep-2 DSPE-PEG-platele t-derived growth factor peptide DSPE-PEG-VCA M-1 targeting peptide DSPE-PEG-angiog enic vessels targeting peptide

Core-shell Nanocapsules Micelles

~180 ~19

PTX and ccombretastatin A4 Flurbiprofen

Polymer hybrid NPs

40-230

Nanocomplexes

15-54

Nanoemulsions

90-120

Micelles

~110

Oxaliplatin, chlorin e6, cisplatin, and siRNA Rhodamine B, NIR664, and gadolinium siRNA, PTX, DTX

Liposomes

~200

Polymeric hybrid NPs Liposomes

DNA, CPT, and rapamycin

HUVECs, 4T1, and A549 cells

References

[42, 70-77, 155, 176, 231-236]

[135] [175] [172, 181, 237] [154, 238]

HUVECs

Cervical carcinoma

[148]

A549

Lung cancer

[78]

PTX

H69 and A549 cells

Lung cancer

[157]

83-101

siRNA

MCF-7 cells

Breast cancer

[79, 80]

~96

Neuroprotectant DOX, PTX, siRNA, miRNA, vinorelbine bitartrate PTX DTX DOX, CPT, PTX, and natural products Calcein, PTX, Rhodamine B, and hydroxychloroquine

BCEC cells

Ischemic stroke Fibrosarcoma, brest cancer, glioblastoma, and breast cancer Gioma Liver cancer

Liposomes

95-120

Micelles Nanosuspensions Polymeric Hybrid NPs

~54 ~100 90-300

U87MG, HT-1080, MCF-7, and TNBC cells C6 cells HepG2 4T1, U87, bEnd.3, U87MG, and HUVECs

Breast cancer and glioma

[239] [88, 89, 92, 93, 96, 240] [90] [241] [91, 94, 95, 105]

HepG2, B16F10, and LO2 cells

Liver cancer and metastatic melanoma

[97, 242]

Lidocaine

-

Pain threshold

[243]

94-104

DOX

MDA-MB-231, MCF-7, and MDA-MB-435S

Breast cancer

[156]

Liposomes

30-123

Rhodamine B and DOX

SMMC-7721, H1299, 4T1, Hep-2, A549, and K562 cells

Liver cancer and lung cancer

[98, 99, 147]

Micelles

~20

PTX

MCF-7 cells

Breast cancer

[173]

Liposomes

~109

DOX

BMECs

Parkinson’s disease

[83]

Liposomes

16-114

BODIPY and Tc99m-HMPAO

MCF-7 cells

Breast cancers

[106, 244]

Liposomes

~95

DOX

-

Prostate cancer

[245]

Liposomes

~56

HRP and AMB

BMECs

BBB

[81, 82]

Liposomes

94-200

DOX and siRNA

SCLC, HMEC-1, and H69 cells

-

[32, 100]

Liposomes

~100

DOX

NCI-H446 and MCF7 cells

Lung cancer

[41]

Hybrid NPs

~185

Cabazitaxe

LNCaP cells

Prostate cancer

[246]

H446 and MCF-7 cells MCF-7 and MDA-MB-231 cells

Liposomes

~100

Liposomes

~158

Liposomes

Micelles Polymer hybrid NPs

~14

PTX

~188

MTX

Micelles

50-400

Curcumin-6 and DiR

Micelles

-

Micelles Micelles

Liposomes

Lung cancer

[133]

Breast cancer

[247]

-

Spinal cord injury

[163]

Coumarin-6 and DiR

bEnd.3 and U87 cells

Glioblastoma

[248]

~12

Temozolomide

U87 and LN229 cells

Glioblastoma

[249]

10-17

Cy7 and gadolinium

HUVEC and MAEC cells as well as fibrin-containing clots

Atherosclerotic plaques

[160, 161]

CisPt, ADM, and DOX

SPC-A1, H1299, SMMC-7721, HUVECs, Colon 26, NL-17, MDA-MA-231, and MDA-MB-435S cells

Colon cancer

[250-252]

100-164

47

DSPE-PEG-low density lipoprotein receptor targeting peptide DSPE-PEG-interle ukin-4 receptor bonding peptide DSPE-PEG-stratu m corneum penetrating peptides DSPE-PEG-antimi crobial peptide DSPE-PEG-oligop eptide of eight aspartate residues

Journal Pre-proof Liposomes

~100

Daunorubicin

THP-1 and NB4 cells

Acute myeloid leukemia

[146]

Liposomes

-

Cytotoxic T lymphocytes

B16F10 cells

Melanoma

[174]

Liposomes

~105

Vemurafenib

A375, B16F10, and HUVECs

Melanoma

[253]

Micelles

~20

Antimicrobial peptide

HUVECs

Sepsis

[177]

Liposomes

~138

Icaritin

-

Osteoporosis

[182]

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Table 3. Nanodiagnostics and nanotherapies decorated with phospholipid-PEG-protein conjugates. Phospholipid-PEG-protei n conjugates

Nanovehicles

Size (nm)

Payloads 125I,

DSPE-PEG-transferrin DSPE-PEG-lactoferrin DSPE-PEG-ephrin-A1 protein DSPE-PEG-P-selectin DSPE-PEG-APTEDB DSPE-PEG-thrombomod ulin

Cells/tissues

In vivo applications

References

HCT-8, Colon 26, HepG2, and K562 cells

Colon cancer and hepatocellular carcinoma

[84-86, 187]

A549 cells HepG2 cells

Lung cancer Hepatocellular carcinoma

[183] [87] [46]

Liposomes

70-441

Micelles Liposomes

~190 ~159

DOX, ODNs, and artmisinin DNA Coumarin-6

Liposomes

~100

MicroRNA

NSCLC cells

-

Nanocomplexes

~149

siRNA

[101]

100-130

DOX

Glioma

[102, 103]

Inorganic hybrid NPs

~42

Fe3O4

HL60 cells U87MG, SCC-7, GL26, and NIH3T3 cells LLC and LNCaP cells

-

Liposomes

Lewis lung carcinoma

[104]

Liposomes

~130

Thrombomodulin

-

Thrombus

[43]

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Table 4. Nanomedicines peripherally decorated with phospholipid-PEG-antibody conjugates. Phospholipids-PEGantibody conjugates

DSPE-PEG-anti-EG FR Fab'

DSPE-PEG-cetuxim ab DSPE-PEG-anti-HE R2 Fab' DOPE-PEG-anti-HE R2 Fab' DSPE-PEG-trastuzu mab DSPE-PEG-anti-VC AM-1 Ab DSPE-PEG-anti-8D 3 mAb DSPE-PEG-scFv DSPE-PEG-anti-IC AM-1 mAb DSPE-PEG-anti-AC 133mAb DSPE-PEG-anti-VE GFR-2 Ab DSPE-PEG-anti-CD 147 mAb DSPE-PEG-HuCC4 9-Fab' DSPE-PEG-anti-hu man glypican-3 Ab DSPE-PEG-anti-LO X1 Ab DPPE-PEG-anti-CE A Ab DSPE-PEG-anti-CD 19 mAb DSPE-PEG-anti-tum or-associated glycoprotein-72 Ab DSPE-PEG-anti CD123 Ab DSPE-PEG-antiCD1 33 mAb DSPE-PEG-insulin-l ike growth factor-I receptor mAb DSPE-PEG-anti-CD 11c Ab DSPE-PEG-anti-plec tin-1 mAb

Nanovehicles

Size (nm)

Payloads

Liposomes

93-246

DOX, MTX, siRNA, WSBA, and dye

Cells/tissues DLD1, HCT116, LS174T, HT29, SW480, LS180, Caco2, Colo205, HCC827, H23, A431, MCF-7, MDA-MB-468, MDAMB-231, and SKBR3 cells MDA-MB-468 cells

DTX ADM and vitamin E acetate

In vivo applications

References

Breast cancer

[33, 144, 254-256]

Micelles

23

Polymeric hybrid NPs

100-246

Polymeric hybrid NPs

~116

Dye

HCC827 and H23 cells

-

[113]

Liposomes

100-194

Pseudomonas exotoxin A

MDA-MB-231, MCF-7, and HeLa cells

-

[111]

Liposomes

-

DNA

SK-BR3, MCF-7, and HeLa cells

-

[110]

Liposomes

~100

Boron

SK-BR-3 cells

-

[257]

SMMC-7721 cells

Breast cancer Hepatocellular carcinoma

[108] [109, 158]

Liposomes

80-132

siRNA and rapamycin DNA

Polymeric hybrid NPs

~132

DNA

SMMC-7721 cells

Liposomes Polymeric hybrid NPs

100-120 ~193

DOX rMETase

PC3, Du-145, LNCaP, and SKBR3 cells SGC-7901 cells

Hepatocellular carcinoma Gastric carcinoma

Liposomes

~105

DOX

A375SM, C32

-

[112]

Liposomes

126

-

Caco-2

-

[263]

Liposomes

~169

DTX

HepG2, A549, and B16F10 cells

Melanoma

[38]

Liver cancer

[264]

Liposomes

138-200

HUVECs

-

[34, 258]

N2A cells

[259] [261, 262] [262]

[260]

Liposomes

~90

DOX

HepG2, Huh-7, PLC, Hep3B, MHCC97L, MHCC97H, HCCLM3, and SMMC-7721 cells

Liposomes

~120

DNA

LS174T, WiDr, and MCF-7 cells

Colorectal adenocarcinoma

[265]

Liposomes

44

siRNA

HepG2, RAW264.7, and CV-1 cells

-

[266]

Liposomes

~145

Fasudil

Carotid artery lesions

Carotid artery lesions

[186]

MKN-45 cells

Gastric cancer

[267]

BxPC3 cells

-

[37]

(3H)

Liposomes

100-130

Polymeric hybrid NPs

~95[264]

Cholesteryl hexadecylether PTX

Liposomes

~120

DOX

Namalwa cells

Health mice

[268]

Liposomes

<100

DNA

LS174T and WiDr cells

Colon carcinoma

[269]

Polymeric hybrid NPs

∼210

siRNA

KG1 and K562 cells

-

[270]

Liposomes

~203

Temozolomide

U87MG and GSCs cells

Glioblastoma

[271]

oxSWNHs

~210

Vincristine

MCF-7 cells

Breast cancer

[107]

Nanocages

Thickness of 5.5 nm

Antigen peptide TRP2

Dendritic cells

Melanoma

[137]

Inorganic hybrid NPs

55-84

Fe3O4

MIAPaCa2 and XPA-1 cells

Pancreatic tumor

[159]

50

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Fig. 1. Different nanotherapies decorated with phospholipid-PEG-ligand conjugates. (A) Liposomes; (B) Micelles; (C) Nanoemulsions; (D) Polymeric hybrid NPs; (E) Inorganic hybrid NPs; (F) Nanocomplexes.

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Fig. 2. Engineering of different targeting phospholipid-PEG conjugates. (A) Synthesis of PEGylated phospholipids with varied reactive groups. (B) Targeting moieties with different functional groups and strategies for the synthesis of targeting phospholipid-PEG conjugates.

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Fig. 3. Different strategies for synthesis of phospholipid-PEG-FA conjugates. (A) Direct amidation strategy. (B) Coupling of phospholipid-SA with NH2-PEG-FA. (C-D) Thiol-maleimide coupling strategy. (E) Direct EDC/NHS coupling strategy. (F) Post-insertion by EDC/NHS coupling strategy.

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Fig. 4. Synthesis of phospholipid-PEG-RGD conjugates. (A) Thiol-maleimide coupling strategy. (B) EDC/NHS coupling strategy. (C) Click chemistry strategy. (D) Post-insertion by EDC/NHS coupling strategy. (E) Post-insertion strategy with DMTMM as a catalyst.

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Fig. 5. Different peptides and proteins conjugated onto PEGylated phospholipids.

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Fig. 6. Synthesis of phospholipid-PEG-antibody conjugates. (A) EDC/NHS coupling strategy. (B) Cyanuric chloride as a linker. (C) Thiol-maleimide coupling strategy using SATA as a linker. (D) Post-insertion strategy using SPDP as a linker. (E) Post-insertion strategy using 2-iminothiolane. (F) Post-insertion strategy with bis(sulfosuccinimidyl) suberate as a linker. (G) Post-insertion strategy by EDC/NHS coupling chemistry. (H) Post-insertion strategy using SMPB as a linker. (I) Post-insertion strategy by thiol-maleimide coupling chemistry. (J) Incorporation of the sulfhydryl group via a redox reaction. 56

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Fig. 7. In vivo tumor imaging in H22 tumor-bearing mice by DSPE-PEG-FA conjugate-modified DiR-MMC-phytosomes or DSPE-PEG-modified DiR-MMC-phytosomes. (A) In vivo fluorescence imaging at 6 h post-injection. The anatomical site indicated is the tumor site. (B) Ex vivo fluorescence imaging of the tumor and organs at 6 h post-injection. (C) Ex vivo distribution in the frozen sections of tumor tissues: (i) bright field, (ii) DiR, and (iii) merge of DiR and bright field images. (D) Quantitative analysis of tumor targeting capacity. *p < 0.05. Reproduced with permission [149]. Copyright 2014, American Chemical Society.

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Fig. 8. In vivo imaging of H446 tumor-bearing mice by DiD-labeled micelles decorated with DSPE-PEG-lanreotide conjugate. (A) In vivo images at different time points after injection. The tumor site is indicated by the white arrow. (B) Fluorescence signals in tumors at different times based on in vivo NIR images. (C) Ex vivo images of tumors and organs excised from H446 tumor-bearing mice at 24 h after administration. (D) Quantified fluorescence signals in tumors and organs based on the ex vivo images. PM-DiD, DiD-loaded micelles; Blank lanreotide-PM + Lanreolide-PM-DiD, lanreotide-modified blank micelles and lanreotide-modified DiD/micelles; Lanreotide-PM-DiD, DSPE-PEG-lanreotide conjugate modified DiD/micelles. Data represent mean ± SD (n = 3). *p  0.05 versus lanreotide-PM-DiD. Reproduced with permission [133]. Copyright 2015, Taylor & Francis.

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Fig. 9. Cy7-labeled micelles modified with DSPE-PEG-VCAM conjugate for in vivo imaging of atherosclerotic plaques in ApoE -/- mice. (A) In vivo real-time imaging of early or mid-stage plaques. i-iv: DSPE-PEG modified Cy7-labeled micelles did not show a strong signal in the aorta at 24 h post injection. Micelles were observed predominately in the bladder and liver. v-vii: NIR in vivo imaging showed the presence of DSPE-PEG-VCAM conjugate-modified Cy7-labeled micelles in the cardiovascular system (denoted by arrow). Either DSPE-PEG modified Cy7-labeled micelles (viii-x) or DSPE-PEG-VCAM conjugate-modified Cy7-labeled micelles (xi-xiii) showed a strong signal in the cardiovascular system at 24 h post injection in the mid-stage plaques. (B) In vivo imaging with targeting or non-targeting Cy7-labeled micelles showed accumulation in the aortic tree. (C) Quantification of the deposition of micelles in the aortic tree showed statistical significance between the control PEG micelles and active targeting VCAM-1 micelles in ApoE-/- mice with early stage plaques (*p < 0.001) and mid-stage plaques (**p < 0.01). Reproduced with permission [160]. Copyright 2014, Elsevier.

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Fig. 10. DSPE-PEG-Apamin conjugate-modified micelles labeled with DiR for in vivo imaging of spinal cord injury. (A) In vivo fluorescent images of five groups of mice treated by DiR-loaded formulations administered via tail vein at 0.5, 1, 2, 4, 8, 12, and 24 h post injection. (B) Ex vivo fluorescent images of brains, spinal cords, and other major organs dissected from animals of the five treatment groups at the same time points post injection. Animals were administered DiR (i), DiR/micelles (ii), DSPE-PEG-Apamin conjugate-modified DiR/micelles pretreated with apamin 1 h prior to the injection (iii), DSPE-PEG-Apamin conjugate-modified DiR/micelles pretreated with dequalinum chloride 1 h prior to the injection (iv), and DSPE-PEG-Apamin conjugate-modified DiR/micelles (v). (C) Ex vivo fluorescent images of brains, spinal cords, and other major organs dissected from spinal cord injury animals and normal mice administered drug-loaded targeting or non-targeting DiR/micelles via tail vein injection. Normal mice were administered DSPE-PEG-Apamin conjugate-modified DiR/micelles (vi) and non-targeting DiR/micelles (vii). Spinal cord injury mice administered non-targeting DiR/micelles (viii) and DSPE-PEG-Apamin conjugate-modified DiR/micelles (ix). Reproduced with permission [163]. Copyright 2014, American Chemical Society.

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Fig. 11. DSPE-PEG-alendronate conjugate-modified nanotherapies for treatment of myelodysplastic syndrome. (A) Fabrication of bone-targeting NPs (BTNPs). (B) In vivo imaging showed BTNPs distribution compared to non-targeting NPs, fluorescent control, and PBS (at 24 h post i.v. injection). (C) Tissue biodistribution of BTNPs in MDS mice after 12 h of administration. Data represent mean ± SEM (n = 4). i, Arsenic trioxide (ATO); ii, Decitabine (DAC). (D) Therapeutic efficacy of BTNPs in a MDS mouse model. iii-v, Serial complete blood count measurements from MDS mice after treatment with DAC/ATO (Red line represents the threshold for normal WBC, PLT, and RBC count). Data represent mean ± SEM (n = 16). vi, Survival curves showing progression to death in different treated groups of mice. Data collected from 3 separate experiments. vii-x, Representative peripheral blood smears from MDS mice after treatment. Reproduced with permission [63]. Copyright 2017, Elsevier.

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Fig. 12. DSPE-PEG-peptide conjugate-modified nanomedicines for cancer treatment. (A) DSPE-PEG-iRGD-modified NPs loaded with AC (Chlorin e6) for treatment of orthotopic 4T1 tumor-bearing mice. i, Fabrication of DSPE-PEG-iRGD-modified NPs; ii, In vivo antitumor effects of NPs. Oxa, oxaliplatin; HOT, hexadecyl-oxaliplatintrimethyleneamine; AC/NPs, Chlorin e6-loaded non-targeted NPs; Targeted AC/NPs, DSPE-PEG-iRGD-modified NPs loading AC. Reproduced with permission [154]. Copyright 2016, Wiley-VCH. (B) DSPE-PEG-α-conotoxin Iml conjugate-modified micelles loading PTX for treatment of MCF-7 tumor-bearing mice. i, Preparation of DSPE-PEG-α-conotoxin Iml conjugate-modified PTX/micelles; ii, Tumor volumes; iii, Body weight changes of mice during antitumor efficacy study. *p < 0.05, vs PBS; **p < 0.01, vs PBS; △p < 0.01, vs Taxol; ☆p < 0.01, vs PM-PTX (PTX/micelles). Reproduced with permission [173]. Copyright 2015, Elsevier. (C) DOX/liposomes decorated with DSPE-PEG-tumor metastasis targeting peptide conjugate for treatment of MDA-MB-231 tumor-bearing mice. i, Preparation of DOX/Liposomes modified with DSPE-PEG-tumor metastasis targeting peptide conjugate. ii, Antitumor activity of DOX/liposomes modified with DSPE-PEG-tumor metastasis targeting peptide conjugate. Relative tumor volume represents the ratio of tumor volume/primary tumor volume. Arrows denote drug administration. *p < 0.05 and **p < 0.01 for targeted DOX/liposomes (TMT-LS-DOX) sample and all other groups. iii, Body weight changes. LS-DOX, DOX-loaded liposomes; TMT-LS-DOX, targeted DOX-loaded liposomes. Reproduced with permission [156]. Copyright 2012, Elsevier. (D) Cytotoxic T lymphocytes (CTLs) coated with DOPE-PEG-interleukin-4 receptor targeting peptide conjugate for treatment of melanoma-bearing mice. i, Preparation of DOPE-PEG-interleukin-4 receptor targeting peptide conjugate coated CTLs. ii, Tumor volumes. iii, Survival rate. IL4RPep-1CTLs represent DOPE-PEG-interleukin-4 receptor targeting peptide conjugate coated CTLs. *p < 0.05 and ***p < 0.001 by one-way analysis of variance; n.s., no significance. Reproduced with permission [174]. Copyright 2018, Elsevier.

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Fig. 13. DSPE-PEG-RGD conjugate functionalized micelles encapsulating flurbiprofen for treatment of ocular inflammation. (A) Fabrication of flurbiprofen-loaded micelles functionalized with DSPE-PEG-RGD conjugate for ocular delivery. (B) Ocular surface retention studies. Fluorescence microscopy of rat eyes after treatment with different coumarin-6 (COU-6) formulations. The white arrows show typical drug retention sites. COU-6-M, coumarin-6 loaded micelles; targeted COU-6-M, DSPE-PEG-RGD-modified micelles loaded with coumarin-6. (C) Clinical symptoms of ocular inflammation such as conjunctiva congestion, swelling, and iris hyperemia were examined using a slit lamp. Fluorescein staining was used for examination of corneal epithelial integrity. White arrowheads indicate the inflammatory sites over the conjunctiva, iris, and cornea. (D) Clinical scores of ocular inflammation in the conjunctiva of rabbit eyes. Statistical analysis of two-way ANOVA with repeated 3 measures was conducted by comparing with the PBS group (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001. (E) Representative optical coherence tomography (OCT) images of corneas and retinas after eyes were treated with the irritant 1% sodium dodecyl sulfate (SDS), 0.03% flurbiprofen sodium (FBNa formulation), flurbiprofen-loaded micelles (M-FBP), or DSPE-PEG-RGD conjugate functionalized micelles containing flurbiprofen (CTFM-FBP). OCT imaging was conducted 24 h after the last instillation. Red arrows show corneal epithelium defects. Double sided arrows indicate the boundaries of the retina. “RPE” indicates the retinal pigment epithelium. The “C” represents the choroid and “S” represents the sclera. Reproduced with permission [175]. Copyright 2018, Wiley-VCH.

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Fig. 14. Micelles functionalized with DSPE-PEG-antimicrobial peptide conjugate for treatment of sepsis. (A) Formation of micelles based on DSPE-PEG-antimicrobial peptide conjugate. (B) Survival curves of mice with cecal ligation and puncture (CLP)-induced sepsis. CLP, untreated CLP-operated mice; KSLW, free KSLW peptide treated mice; PEG5K-KSLW, the PEG5000-KSLW peptide conjugate treated group; PLM-KSLW, mice treated with DSPE-PEG-KSLW peptide conjugate micelles. (C) Bacterial loads in the peritoneal fluid of CLP-operated mice. (D) Bacterial loads in the blood of CLP-operated mice. Various KSLW formulations were intravenously injected into mice with CLP-induced sepsis at 12 h after the CLP procedure. (E-F) Bacterial loads were analyzed by counting bacterial colonies. Reproduced with permission [177]. Copyright 2017, Ivyspring International Publisher.

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Fig. 15. DSPE-PEG-cRGDfK peptide conjugate modified NPs for targeted-treatment of abdominal aortic aneurysms. (A) Schematic illustration of a ROS-responsive rapamycin nanotherapy decorated with a DSPE-PEG and cRGDfK conjugate. (B) Fluorescence images of VSMCs incubated with Cy5-labeled ROS-responsive targeting NPs (ROCy5 NP) or Cy5-labeled ROS-responsive non-targeting NPs (OCy5 NP) for different periods of time. Scale bars, 20 μm. (C) Ex vivo images showing the accumulation of Cy7.5-labeled ROS-responsive targeting NPs (ROCy7.5 NP) or Cy7.5-labeled ROS-responsive non-targeting NPs (OCy7.5 NP) in aneurysmal aortas. (D) Hematoxylin and eosin (H&E), Alizarin Red, or VVG stained histological sections of aneurismal aortas. ROR NP, DSPE-PEG-cRGDfK-modified ROS-responsive NPs encapsulating rapamycin; OR NP, rapamycin-loaded ROS-responsive NPs. Reproduced with permission [181]. Copyright 2018, Elsevier.

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Declaration of interests ■The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: