Amphiphilic Drug Conjugates as Nanomedicines for Combined

Review pubs.acs.org/bc

Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

Amphiphilic Drug Conjugates as Nanomedicines for Combined Cancer Therapy Chuang Gao,† Pravin Bhattarai,† Min Chen,† Nisi Zhang,† Sadaf Hameed,† Xiuli Yue,*,‡ and Zhifei Dai*,† †

Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, 100871, China School of Environment, Harbin Institute of Technology, Harbin 150080, China

Bioconjugate Chem. Downloaded from pubs.acs.org by UNIV OF RHODE ISLAND on 11/29/18. For personal use only.

‡

ABSTRACT: Chemotherapy suffers from some limitations such as poor bioavailability, rapid clearance from blood, poor cellular uptake, low tumor accumulation, severe side effects on healthy tissues and most importantly multidrug resistance (MDR) in cancer cells. Nowadays, a series of smart drug delivery system (DDS) based on amphiphilic drug conjugates (ADCs) has been developed to solve these issues, including polymer−drug conjugate (PDC), phospholipid-mimicking prodrugs, peptide−drug conjugates (PepDCs), pure nanodrug (PND), amphiphilic drug−drug conjugate (ADDC), and Janus drug− drug conjugate (JDDC). These ADCs can self-assemble into nanoparticles (NPs) or microbubbles (MBs) for targeted drug delivery by minimizing the net amount of excipients, realizing great goals, such as stealth behavior and physical integrity, high drug loading content, no premature leakage, long blood circulation time, fixed drug combination, and controlled drug-release kinetics. Besides, these selfassembled systems can be further used to load additional therapeutic agents and imaging contrast agents for combined therapy, personalized monitoring of in vivo tumor targeting, and the pharmacokinetics of drugs for predicting the therapeutic outcome. In this review, we will summarize the latest progress in the development of ADCs based combination chemotherapy and discuss the important roles for overcoming the tumor MDR.

■

chronic condition.5,9−11 One combination approach is to coadminister drugs that work by different molecular mechanisms to afford a synergistic therapeutic effect. This approach particularly benefits therapeutic response by preventing cancer cells from developing a compensatory resistance mechanism like in single or sequentially administered agents.6 Another interesting approach is to use a drug that can particularly inhibit the resistance mechanism of tumors and may “resensitize” the patient to the original treatment.12 Apparently, the effectiveness of this combination strategy rests upon the careful selection of drugs and well-defined delivery methods. For instance, synergism or antagonism of more than one drug at the tumor site often relies on the molar ratio of the combined drugs.7 Irinotecan and fluoropyrimidine derivative floxuridine (FUDR) at a 1:1 molar ratio showed great synergistic efficacy in tumor therapy; however, when the ratio changed to 10:1, the two drugs showed antagonistic efficacy.13 This highlights the importance of the drug ratio in combination chemotherapy to yield better therapeutic outcomes. Therefore, “How to deliver the synergistic drug ratios in vivo?” plays a key role in the translation of combination chemotherapy from the bench to the bedside.

INTRODUCTION Chemotherapy is still a first choice for the treatment of most cancers due to its presumed higher clinical efficiency. However, most of the chemotherapeutic drugs suffer from limitations such as poor bioavailability, rapid clearance from blood, poor cellular uptake, low tumor accumulation, the inability to differentiate healthy and tumor cells, and most importantly the development of multidrug resistance (MDR).1−4 Drug resistance is one of the major challenges in cancer therapy.5−8 One of the possible reasons for intrinsic resistance is the molecular alterations in cancer cells that make them insensitive toward a particular drug before the treatment itself. In an alternative case, few cancer types might adapt to the drugs initially but acquire molecular changes later to escape the drugs‘ effect. In an extreme case, all of these factors might interplay together in the same tumor to make the condition even worse. In relevance to either of the cases, molecular alterations in the cancer cells responsible for MDR are usually accompanied by the mutation of the drug’s target. Such mutation can alter drugs‘ interactions with tumors and the associated microenvironment resulting in a limited chemotherapeutic efficacy. Strategies to address MDR could have a profoundly beneficial impact on cancer treatment. At this time, a large number of preclinical studies have highlighted combination therapy as the best possible way to overcome or delay the development of resistance; therefore cancer could become a © XXXX American Chemical Society

Received: October 1, 2018 Revised: October 28, 2018

A

DOI: 10.1021/acs.bioconjchem.8b00692 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Review

Bioconjugate Chemistry

toxicity metabolic pathway due to the easy disintegration of self-assembled NPs into individual molecules.3,37,41−45 In addition to the aforementioned advantages, ADCs can also be used as a multifunctional nanoplatform for, e.g., loading multiple drugs or therapeutic agents, to afford a synergistic therapeutic effect. In particular, multifunctional ADDC with temporally controlled drug release profile could be an invaluable tool to conquer the tumor heterogeneity and drug resistance issues.41,44,46 Nowadays, a series of smart DDS based on ADCs have been developed, including polymer−drug conjugate, phospholipidmimicking prodrugs, pure nanodrug, ADDC, and Janus drug− drug conjugate (JDDC) (Table 1). Among all of these, JDDC has shown great potential for clinical application due to its ability to self-assemble into not only nanodrugs but also microbubbles (MBs) without using a large amount of the inert carrier materials.46,47 In addition to the drug delivery applications, JDDC MBs can also be used as a powerful contrast agent for perfusion and general tissue delineation in US imaging.48 Most importantly, the JDDC MBs can be delivered to the target tumor tissue under the guidance of realtime ultrasound imaging, also called the ultrasound-targeted microbubble destruction (UTMD) technique.46−48 Besides, multiple types of therapeutic molecules and imaging agents can be simultaneously coloaded into the delivery system for combination cancer theranostics.47,49 In this review, we will summarize the latest progress in the fabrication of various ADCs based combination chemotherapy, highlight the therapeutic efficiency, and critically discuss ideas to overcome the tumor heterogeneity and MDR issues. Polymer−Drug Conjugate. The nanoformulation comprising unique bulk and surface properties are a universal carrier to load free drugs and deliver them to the targeted diseased site such as a tumor, mainly via the EPR effect.18,50,51 However, a majority of chemotherapeutic nanomedicine often fails to demonstrate enhanced efficacy in a human clinical trial because of the drug leakage, low loading efficiency, and overdosage of the inert excipients.37 In the past few years, to overcome limitations associated with the general physical encapsulation technique, drugs are often chemically conjugated to polymers resulting in an amphiphilic PDC that can readily self-assemble into polymeric NPs with improved solubility and minimal toxicity.52 Furthermore, the PDCs can also provide stealth behavior and physical integrity, prevent drugs from rapid systemic clearance, degradation, metabolism while in circulation, and systemic drug exposure in healthy tissues and organs. Importantly, covalent conjugation between drug and drug (vector) can regulate both the drug-release profile and pharmacokinetics in order to improve therapeutic efficacy.34,53 CRLX101 (formerly known as IT-101) is the first de novo PDC to reach the clinic.52,54−56 CRLX101 consists of camptothecin (CPT) covalently conjugated to a linear, cyclodextrin-poly(ethylene glycol) (PEG) copolymer, and self-assembles into NPs. The solubility of CPT increased by approximately 3 orders of magnitude after conjugation to the cyclodextrin-containing polymer via glycine.57 Thanks to the proprietary cyclodextrin polymeric nanoparticle (CDP) technology,83,84 after the CRLX101 entered cells, the lactone form (the active form) of CPT was released slowly due to the cleavage of the ester linker at acidic conditions,57 thus leading to long tumor exposures of the drug and prolonged HIF-1α and topoisomerase I inhibition (target of CPT) in mouse xenografts.55,85 The antitumor activity as observed in vivo

Nanoparticles (NPs)-based anticancer drug delivery systems (DDS) undoubtedly represent a promising technology to fight and cure cancer.14−19 In general, most of the commonly available chemotherapeutic drugs are encapsulated/loaded in the nanosized vehicles including liposomes,20−22 vesicles,23,24 polymeric NPs,25−27 and inorganic NPs.28,29 The unique surface features, e.g., hydrophilic corona, of as-prepared NPs greatly enhance blood circulation time in vivo resulting in the passive accumulation and delivery of payloads at the specific site via so-called enhanced permeability and retention (EPR) effect. Several studies have reported a comparative advantage of NPs-based DDS over free drugs to improve the therapeutic efficacy against most of the tumors including resistant tumors as well.30−32 In 1995, Doxil became the first Food and Drug Administration (FDA)-approved nanodrug for the treatment of cancer. Doxil exhibited a very high remotely loaded DOX, prolonged blood circulation time, and a highly stable “liquid ordered” bilayer comprising phospholipids such as phosphatidylcholine and cholesterol with a high-phase transition temperature (Tm = 53 °C).33 Since then, many other functional nanoscale DDS have been evaluated in preclinical and clinical studies, and some of them have already advanced into clinically approved liposomal drug formulations.1,34 However, this nanosystem barely accounts for the complete tumor eradication and is often accompanied by tumor recurrence or resistance to the therapy with the passage of time. In a recent advancement, multifunctional NPs, for instance, chemotherapy, light-triggered hyperthermia, or photodynamic treatment in a single nanoplatform, have successfully demonstrated maximum therapeutic benefit. Despite rapid progress in the field, what is still unclear is the type of combination approach that would inherently enhance the therapeutic efficacy without increasing complexity in the clinical translation process of multifunctional NPs. Toward this goal, NPs delivering synergistic drug ratios in vivo and enhancing delivery to a tumor for combination chemotherapy have recently been of immense interest. With regard to this, more efforts are being paid to accelerating clinical approval of DDS for combination therapy. In 2017, the USFDA approved Vyxeos for the treatment of adults diagnosed with two types of acute myeloid leukemia (AML): myelodysplasia-related changes (AML-MRC) or newly diagnosed therapy-related AML (t-AML).35,36 Vyxeos liposome became the first approved combination nanomedicine in the clinic, which is a fixed combination of the chemotherapeutic drugs daunorubicin and cytarabine. However, combination nanomedicine has not shown the full potential yet because of few limitations such as drug leakage, low drug loading efficiency, and overdosage of the inert vector.37 In addition, most of the aforementioned DDSs are usually a multicompartment system and are fabricated after combining several individual components to form a nanomedicine. The interdependence of each individual component make such a system even more challenging and therefore results in the inconsistent and unpredictable formulation outcomes.38 To address such issues, more effort has been devoted to designing nanomedicines using drug molecules, such as amphiphilic drug conjugates (ADCs).4,39,40 The amphiphilic drugs themselves take part in the self-assembly process and therefore exclude the need for any additional vectors/ excipients. Notably, ADCs have shown improved anticancer efficacy in multiple ways, such as (a) improved drug loading capacity, (b) better physicochemical properties, and (c) a lowB


C

Amphiphilic drug conjugate

Peptide−drug conjugate

Phospholipidmimicking prodrug

Pure nanodrug

Polymer−drug conjugate

ADC types

Cisplatin+Vorinostat

CPT+Ara-C

MTX+CPT

GT+CPT

CA4+Ir

FUDR+CPT

HCPT-peptide and Cisplatin HCPT-DTDEPEG FdU+BdM

F3-ELP-C8DOX LHRH-ELPC8-DOX

di-CPT-OEG

di-CPT-GPC

Ursolic acid

DOX-s-s-DOX

PTX-s-s-PTX DOX-s-s-DOX

PTX-acetalPEG PTX dimers

CPT-FA

PEG-bPMPMC-gPTX DOX-LAC

SN-38-R

Drug regimen

HeLa tumor-bearing nude mice B16F10 tumor bearing mice A549/DR tumor-bearing mice

MCF-7, HUVECs, and CSCs HeLa, MCF-7

MCF-7, HeLa, and MCF-7/ADR cells HT-29

A549

C26 tumor bearing mice doxorubicin-resistant breast cancer mouse model A549/DDP

MCF-7 tumor bearing mice A549 cancer xenograft growth model MCF-7 tumor bearing mice MCF-7 and SKOV-3

HeLa or B16F10 tumor bearing nude mice A-549 MCF-7, MCF-7/ADR

HeLa, MCF-7, KB and A549 HeLa, MDA-MB-231

Carrier free stimuli-responsive hybrid prodrug conjugate, controlled drug release, precise drug loading as high as ∼75 wt %, a prominent synergistic effect on HeLa and MCF-7 cancer cells. A precise drug-to-drug ratio, pH-/esterase-responsive drug release, real-time monitoring fluorescence “Off−On” switch, codeliver multidrug to different sites of action with distinct anticancer mechanisms and kill folate receptor-overexpressing tumor cells in a synergistic way. A redox-sensitive drug−drug conjugate, improved water solubility of CPT and the cell membrane permeability of Ara-C, rapidly internalized by tumor cells, minimal injury to normal cells, simultaneous release of drugs improve their anticancer activity and afford synergistic chemotherapy effects. Prolonged blood circulation and increased accumulation at the tumor site, enhanced cellular uptake and DNA binding efficacy, decreased intracellular GSH and downregulation of cisplatin resistance-related proteins MRP1 and BCL-2, sufficiently intracellular drug release, significant anticancer efficacy toward cisplatin resistance nonsmall cell lung cancer (NSCLC).

Self-assembled NPs from CPT-FUDR, simultaneously release drugs at a fixed dosage, synergistic combination chemotherapy including improved cell apoptosis, varied cell cycle arrest, as well as effective inhibition of cancer cell proliferation. Simultaneously inhibiting differentiated cancer cells, CSCs and endothelial cells under a normoxic environment

Enabling codelivery of two drugs at a high and fixed content, and reversing resistant cells depicting enhanced anticancer activity.

Supramolecular nanomedicines for nuclear delivery of dual synergistic anticancer drugs, improved inhibition capacity to resistant cancer cells, and promote the synergistic tumor suppression property in vivo. Precisely controlled drug loading content and GSH-triggered degradation for enhanced anticancer drug delivery.

̈ Undergoes assembly to form liposomes without any excipient, high loading content of CPT, sustainable release of free CPT, and comparable cytotoxicity to the naive drug form against MCF-7. Amphiphilic phospholipid-mimicking nanocapsules with a high CPT loading, no burst release, load additional hydrophilic DOX with high loading efficiency, and show enhanced antitumor activity. Tumor-homing and pH-responsive polypeptide−doxorubicin nanoparticles, F3 peptide that can specifically bind to nucleolin which is highly expressed on the membrane of tumor cells. Tumor-homing, pH- and ultrasound-responsive nanoconjugates, accelerated cellular uptake and enhanced cytotoxicity to DOX-resistant cancer cells when exposed to US.

Carrier-free nanodrug enhancing anticancer efficacy, proliferation inhibition and apoptosis induction against cancer cells, and potential immune function.

High drug loading, redox-responsive releasing behavior of PTX, capable of loading photothermal agent (DiR), and execute synergetic chemo-photothermal therapy. Reduction responsive drug−drug conjugate linked through a disulfide bond via a simple one step reaction, significantly higher cellular uptake through clathrin-mediated endocytosis pathway and subsequent antitumor activity. The length of linker could influence self-assembly process, and linker types and linkage site could affect antitumor efficacy.

Excellent targeting ability and long blood circulation time, high drug loading ratio, on-demand drug release behavior, enhanced anticancer activity and minimum side effects. GSH-responsive small prodrug system, high and precise drug loading, “switchable” fluorescence in response to GSH, and higher cytotoxicity for FA receptor positive KB tumor cells Endosomal pH-responsive micelles, high PTX loading combining both physical entrapment and chemical conjugation, and programmed drug release behavior for enhanced anticancer effect. High solubility regardless of any surfactants or adjuvants (2500 folds than free PTX), possess effective cellular uptake, reduced systemic toxicity and enhanced antitumor efficacy toward human cervical tumor.

SMMC 7721

Outcomes Solvent- and adjuvant-free delivery platform with excellent stability to survive in the blood, facilitate sufficient delivery of the drugs to target tumor sites via EPR effects, and modular platform to attach targeting ligand via coassembly. Reduction-sensitive polymeric PTX prodrug that can form micelles or polymersomes, encapsulate both hydrophilic/hydrophobic DOX, and trigger synergistic effect against drug-resistant cells.

HT-29, HC-116, and A549 Hela, MCF-7/ADR

Cancer type

Table 1. List of ADC in Cancer Therapy

78

77

76

75

74

73

72

71

70

69

68

37

67

66

65

64 39

63

62

61

60

59

58

refs

Bioconjugate Chemistry Review


Review

81 82

80

Transformable prodrug amphiphiles prolongs blood circulation significantly, high solubility and stability, release intact CPT in a redox-responsive manner, exhibits potent cytotoxicity as a parent CPT, and efficient tumor accumulation for excellent therapeutic efficacy. Synergistic killing of the cancer cells but also reduce the undesirable side effects on normal cells. Self-delivering, adjustable feed ratio, synchronous release of three drugs, and optimal synergistic therapeutic effect.

79

CPT+CB GEM+Cb+Ir

CPT+EB

Cancer type

FLO-1, SKGT-4, and JHU (JHU-EsoAd1) cells HCT116 tumor-bearing mice MRC-5, MCF-7 A-549

Drug regimen

Camptothecin +Capecitabine

Outcomes

greatly surpassed the therapeutic efficiency of two similar FDAapproved analogues of CPT, irinotecan, and topotecan.55,57,85,86 Interestingly, the low molecular weight polymeric remnants after complete release of CPT were cleared easily via the kidney, which is an important prerequisite for clinical translation. The conjugation of linear chains of PEG on the NP surfaces (PEGylation) significantly improves the pharmacokinetic and pharmacodynamic properties of the nanomedicine.87 The functional PEGylated corona prevents drug accumulation in reticuloendothelial system (RES) organs but simultaneously enhances drug accumulation in the tumor site.88 PEG can also be conjugated to the hydrophobic drugs to formulate prodrug nanomedicines. These prodrugs can self-assemble into micelles and ultimately release the drug based on pH or enzymatic activation at the targeted site. For instance, NKTR-102,89 NKTR-105, 90 and EZN-2208 91 are PEG prodrugs of irinotecan, docetaxel, and SN-38, respectively, that are currently used in a clinical trial for the treatment of patients with solid tumor malignancies. Alternatively, poly(glutamic acid) can also be used to develop similar polymeric prodrugs. For example, Opaxio,92 a poly(glutamic acid) conjugated paclitaxel prodrug, release drug after reacting with enzyme cathepsin B in the tumor cell and is currently in Phase 3 clinical trial for the treatment of ovarian cancer. The other similar formulation in clinical trial combines carboplatin for the treatment of nonsmall cell lung cancer. Phospholipid-Mimicking Prodrugs. In comparison to several nanoformulations, liposomal nanocarriers are already well-established in clinics and the majority of those in preclinical studies have the potential to augment therapeutic efficacy. Beside a simple fabrication technique, liposomal nanocarriers are more versatile and can accommodate multiple drugs for coadministration while simultaneously increasing the bioavailability of encapsulated drugs in tumors and minimizing direct cytotoxicity in normal tissues. However, low drug loading capacity and use of a large number of excipients in the preparation of liposome severely limits the wide application of this nanomedicine. As compared to the proportion of therapeutic drug in nanoformulation which is generally less than 10%, a large amount of delivered excipients have several drawbacks. For instance, a larger amount of phospholipids in the blood results in increased systemic lipotoxicity, and after the release of drug these excipients should be excreted instantaneously which might impose an unnecessary burden on the excretion mechanism. Although continuous efforts are placed to resolve such issues, exemplary groundwork by Shen et al.37 seems very effective and practical to implement in future works. The idea capitalizes on the use of hydrophobic drugs to replace the hydrophobic part of a lipid bilayer. This technique not only minimizes the net amount of phospholipids in DDS but also improves the loading content for various hydrophobic drugs. In a proof-of-concept, the authors successfully modified an extremely hydrophobic, yet potent, chemotherapeutic drug, CPT (has a very low water solubility of about 3 μg/mL) to replace the fatty acid(s) in phospholipids. The modified amphiphilic phospholipid-mimic prodrug comprising a very short nonionic ethylene glycol (OEG) chain with only eight repeating units and the drug itself as a hydrophilic and hydrophobic counterpart, respectively (Figure 1, OEG-CPT and OEG-DiCPT). Here, the OEG chain greatly helped to lower the critical vesicle formation concentration of nanocapsules (NCs) and also improve the

ADC types

Table 1. continued

Represents a conceptual advancement in integrating two structurally distinct drugs having different action mechanisms into a single DDS to construct self-deliverable nanomedicines for more effective combination chemotherapy against esophageal cancer cell lines.

refs


D


Review


the tumor cell surface. In a recent advancement, PepDCs have been extensively utilized to synthesize self-assembling drug conjugates that have unique advantages in relevance to nanoperspective to attain higher therapeutic efficiency during cancer treatment. By taking advantage of one-component nanomedicine, this strategy usually allows facile synthesis of PepDCs without need of any excipients. The design basically utilizes the hydrophilic property of peptide sequence that is attached to a hydrophobic anticancer drug, prodrug assembly, via a cleavable linker, and the attached peptide can help further stabilize the nanocarrier in an aqueous medium and therefore function for both assembly and targeting purposes. Such PepDCs can break down over time or in the presence of specific stimuli inside tumor microenvironment to release and activate the attached drug moiety. The advantages of PepDCs in cancer treatment specially encompasses improvement in the solubility of extremely hydrophobic drugs, higher drug loading, and most importantly enhanced internalization of nanomedicines imbued with surface peptides thereby resulting in improved therapeutic efficacy at the targeted sites. The indepth discussion related to such peptides has already been extensively reviewed elsewhere.96 With regard to the improvement in uptake efficiency of PepDCs, Wang et al. has recently designed a tumor-homing and pH-responsive polypeptide (F3ELP-C8) containing three parts: tumor-homing peptide F3, elastin-like polypeptide (ELP) and cysteine-rich segment (GlyGly-Cys)8 (C8).68 Then, the acid-labile hydrazone linker to link the DOX and polypeptide to form PepDCs (F3-ELP-C8DOX). The PepDCs could self-assemble in an aqueous solution to yield NCs. After injection into mice with a murine cancer model, the F3-ELP-C8-DOX nanodrugs showed 4.2fold and 1.8-fold higher drug concentration compared with free DOX and ELP-C8-DOX. This is mainly because of the internalization of F3 peptide that can specifically bind to nucleolin which is highly expressed on the membrane of tumor endothelial cells and tumor cells. Furthermore, the free DOX were quickly released due to the hydrolyzation of hydrazone linker under acid pH condition resulting in higher drug concentration that can later trigger significant antitumor efficacy. Furthermore, the diversity of amino acid combinations69 and linker molecular97 enables the facile preparation of many different PepDCs, showing a great potenitial for targeted drug delivery. However, careful selection of these components during the design of PepDCs is vital for determining the best drug delivery approaches. Pure Nanodrug (PND). Beside phospholipid-mimicking prodrug nanoassembly, another class of NPs known as a PND has also been developed. PND is a kind of carrier-free DDS composed of several pharmaceutically active drugs with enhanced synergistic anticancer efficiency.23,41,45,98 One of the common problems in the design and clinical application of the nanomedicine is the extreme hydrophobicity of chemotherapeutic drugs. So far, the use of various solubilizing surfactants and few chemical modifications have surpassed the solubility issues to some extent. Nonetheless, the use of additional excipients as surfactants still limits the clinical efficacy or translational ability of these clinically potent drugs. It is commendable to avoid such fabrication techniques to yield better clinical outcomes. One of the best alternatives is to make use of active pharmaceutical ingredients themselves as a surfactant. For instance, DOX composed of hydrophobic anthracycline rings and a hydrophobic ring system with abundant hydroxyl groups adjacent to the amino sugar nearly

Figure 1. Amphiphilic camptothecin (CPT) prodrugs (OEG-CPT and OEG-DiCPT) and their self-assembly into nanocapsules (NCs) to load other drugs, such as DOX (hydrophobic membrane is solely made of CPT moieties). Adapted with permission from ref 37. Copyright 2010 American Chemical Society.

drugs‘ solubility. The prodrug conjugated by a thioester bond could self-assemble in an aqueous solution to yield NCs and easily hydrolyze in the presence of esterase. To enhance the therapeutic ability of the NC, additional hydrophilic DOX was loaded at a high loading efficiency. Such a multifunctional NC further resolved two important aspects: (1) higher stability and loading of CPT (58 wt %) with no burst release, and (2) synergistic combination therapy by the release of a higher amount of DOX. A similar strategy can also be used to replace the hydrophilic head of phospholipid with the water-soluble drug, forming another kind of phospholipid-mimicking prodrug. In an example, Feng et al.45 utilized a phospholipid conjugated cisplatin-prodrug (Pt(IV)-DSPE) and other commercially available phospholipids to self-assemble into liposomes. A near-infrared fluorescent dye, 1,1′-dioctadecyl3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) was incorporated into the liposomes to facilitate bimodal imaging-guided photothermal-chemotherapy. The results demonstrated finely tunable drug compositions, good uptake in tumors, and the ability to load multifunctional therapeutic or imaging agents. However, one major disadvantage of such DDS is the inability to control the molar ratio of multiple drugs resulting in compromised therapeutic efficacy. Peptide−Drug Conjugates (PepDCs). Peptide−drug conjugates (PepDCs) are an emerging class of prodrugs comprising three main entities: drug, linker, and peptide sequence and have emerged as a good choice specially for the drug delivery applications.93 The simple PepDCs are often formed through the covalent bonding of a peptide sequence to a drug via a cleavable linker. This strategy can also be easily exploited to yield self-assembled nanomedicines and examine the biological activities of several potent medicinal drugs for the improvement of treatment efficacy. Despite the huge market and overwhelming success of antibody-conjugated drug conjugates, PepDCs provide a more simple and economically viable route for drug delivery and targeting purposes. The major advantage of short peptide decorated prodrugs allows facile delivery of nanomedicines, which is usually diffcult to achieve in antibody-conjugated drug types. In addition, the synthesis of peptides are economically viable and can overcome high production rates of antibody-conjugated drugs, are easily biodegradable, and do not elicit any immunogenic responses in vivo.94 Thanks to the solid-phase peptide synthesis (SPPS) technique95 that allows screening of multiple amino acid sequence to take control over the physicochemical properties of the conjugate and simultaneously allow active targeting to various receptors expressed in E


Review


as compared to the free PNDs. The differences in the cytotoxicity were possibly due to the differential cellular uptake in tumor cell lines and importantly the surface state of PNDs. However, detailed in vivo study is further warranted to understand the role of free and surface modified PND in cancer therapy. It is worth mentioning that Kasai et al. from the same research group had first reported a viable method to downsize PNDs below 100 nm by conjugating two SN-38 drug molecules to form dimers of size 30−50 nm via reprecipitation.100 However, this technique is restricted particularly to specific drug-types and requires further modifications if applied to other drug molecules. At the present time, the common synthesis routes for preparing PNDs encompass the aforementioned conventional reprecipitation methods, and a few other new approaches such as emulsion-templated freezedrying technique,101 nanocrystal technology,102 surfactantstripping,103 and also the anodized aluminum oxide (AAO) template-assisted method.104 Although these routes have been implemented successfully in the preparation of PNDs, few challenges such as stability, size, batch-to-batch variations, low production rates, and the use of inorganic templates still prevail. In this regard, more robust and economically viable synthesis routes are required to realize the full potential of PNDs. To overcome this challenge, Zhang et al. very recently reported a novel green, low-cost, and scalable ice-templateassisted approach for the mass-production of Curcumin (Cur) PNDs by nearly ∼140 times compared to the pre-existing reprecipitation method.105 Most importantly, this technique has also been applied for the preparation of seven clinically viable hydrophobic drugs including PTX, CPT, and MTX. Besides, as-prepared PNDs showed relatively higher serum stability (48 h at 50% FBS, 37 °C) which could be of higher importance for future clinical translation. Amphiphilic Drug − Drug Conjugate. Pure nanodrug can be easily developed by nanoassembly technology; however, the strategy mainly depends on the characteristic structure of the drug and thus limits their wide range of applications. A relatively more versatile approach that can coassemble drugs irrespective of its molecular conformation has evolved recently. For instance, two anticancer drugs having dissimilar chemical properties are ensembled as a single component to yield amphiphilic drug conjugates for synergistic therapy. Inspired by the self-assembly of amphiphilic surfactants and polymers, it can be expected that the amphiphilic drug conjugates comprising one hydrophilic and another hydrophobic drug might also form NPs in a similar fashion. Moreover, since these two drugs have different pharmacokinetics, it is therefore possible that these drugs induce nonoverlapping but synergistic pharmacological effects and simultaneously improve the therapeutic efficacy in vitro/in vivo.42,44,73 In a recent example, Zhang et al.106 reported a novel amphiphilic drug conjugate using two potent anticancer drugs having dissimilar solubility indexes, hydrophilic floxuridine (FdU) and hydrophobic bendamustine (BdM). The two drugs FdU and BdM function independently: the former has a specific activity in DNA but does not affect RNA while the latter is a bifunctional alkylating agent having antimetabolite properties.107,108 The amphiphilic FdU−BdM drugs interconnected by an ester bond could readily self-assemble into stable and uniform NPs in aqueous solution (Figure 3). However, after uptake by tumor cells and excess esterase enzyme activity, the conjugates could be easily disintegrated into individual free drugs in vitro. The excellent anticancer activity of released drugs could easily overcome the

mimics the characteristics of a surfactant. Thus, DOX could also potentially be used as a stabilizing surfactant for nanosizing various drugs. Additionally, DOX itself being a pharmaceutically active component eliminates any limitations owing to the overdose of excipients and can participate more conveniently to load additional drugs resulting in a PND for dual-drug combination therapy. In a recent report by Liang et al.,42 it was possible to create a carrier-free pure PND simply by incorporating two clinically acceptable chemotherapeutic drugs, 10-hydroxycamptothecin (HCPT) and DOX, via a simple reprecipitation method, also referred to as a “green“ method. This method has a competitive advantage over conventional synthesis routes in terms of simplicity, higher efficiency, and eco-friendliness, and most importantly it restricts use of any non-FDA-approved ingredients during the entire synthesis process. As mentioned earlier, DOX’s inherent characteristics as a surfactant circumvented the necessity for excipients, therefore resulting in an excellent aqueous solubility and stability of HCPT. The stability of asprepared PNDs in 10% fetal bovine serum (FBS) was nearly ∼2 h and was verfied using both SEM and fluoresence microscopy while the stability was improved over ∼7 days in an aqueous solution. Besides, the morphology was mostly dependent on the molar ratio of DOX to HCPT varying from a uniform spherical morphology to nanorods and nanospheres (Figure 2). The final optimized spherical PNDs depicted much

Figure 2. a. Coassembly of HCPT and DOX molecules in water. b. SEM images of HCPT/DOX particles assembled from different molar ratios of DOX to HCPT (0.5:1, 2:1, and 4:1). The scale bar is 1 μm. Adapted with permission from ref 42. Copyright 2015 American Chemical Society.

higher intracellular drug retention compared to free drugs because of their enhanced internalization rates and inhibition to P-glycoprotein (P-gp) mediated drug efflux. These results indicated that the self-assembled PND platform could offer new means for enhancing the drug loading content and combating drug resistance in a cancer cell by combination chemotherapy. In another study, Koseki et al.99 evaluated the cellular toxicity of two newly synthesized PND, namely, SN-38 and podophyllotoxin dimer of size around 30−50 nm. Interestingly, the authors revealed a different cytotoxicity index of PND as tested in three different human cancer cell lines: KPL-4, HepG2, and MCF-7. In addition, PND modified with polysorbate 80 coating revealed enhanced cellular toxicity F


Review


ratio; however, it still uses the excipients such as DSPE-lipids thereby compromising the effective drug loading content. Despite simplicity in the fabrication procedure and subsequent efforts in evaluating in vivo biodistribution and pharmacokinetics of ADCs, the clinical translation statistics are extremely disappointing.113 This might possibly be due to the limited control of the synthesis procedure over nanostructure size and morphology. Among all these ADDCs, JDDC has shown great potential for clinical application due to its highly symmetric structure and its layer formimg ability to self-assemble into liposome-like nanocapsules without using a large amount of the inert carrier materials. So far, much effort has been devoted to designing combination nanomedicines that can be delivered simultaneously and act synergistically in vivo so as to overcome the tumor heterogeneity and drug resistance issues.114,115 Celator Pharmaceutials, Inc. (previously; now a subsidiary of Jazz Pharmaceuticals) is a clinical-stage biopharmaceutical company for transforming the science of combination therapy and developing anticancer products.116 Celator’s proprietary technology platform enables the rational design and rapid evaluation of optimized combinations incorporating traditional chemotherapies as well as molecularly targeted agents thought to enhance anticancer activity. Previously, the company successfully conducted the clinical development of a nanoliposomal formulation comprising irinotecan and floxuridine (CPX-1). The CPX-1 formulation is a standard treatment regimen for metastatic colorectal cancer.117,118 The in vivo delivery of irinotecan and floxuridine (FUDR) coencapsulated in a liposome at a molar ratio of 1:1 (CPX-1) demonstrated enhanced therapeutic efficacy compared to both simple mixtures of two drugs in saline or individual liposomal agents.117,118 Nevertheless, liposome-based nanocarriers coencapsulating more than one drug have some major limitations, such as limited drug loading and encapsulation efficiency, stability, optimization of the drug ratio, and controlled release of chemically disparate drugs over time. Most importantly, fabrication of NPs having a perfect biphasic structure to deliver a fixed ratio of drugs (>1) is still a major challenge for scientists.119 To expand the scope of liposome-like DDS and overcome associated disadvantages, Liang et al. recently envisioned an amphiphilic NC from a highly symmetric Janus camptothecin-floxuridine conjugate (JCFC) (Figure 4).46 The liposome-mimicking NC (JCFC) was synthesized by conjugating two hydrophilic and hydrophobic drugs FUDR and CPT, respectively, to a multivalent pentaerythritol via a hydrolyzable ester linkage.120 The as-synthesized amphiphilic JCFC NCs self-assembled in 1:1 ratio of CPT/FUDR resulting in a uniform biphasic structure, also known as Janus (Figure 5). The in vitro and in vivo results depicted very high drug loading content, no premature release, long blood retention time, and enhanced tumor accumulation via EPR effect. The high loading and stability were due to the use of the drug−drug conjugate as a major ingredient in the formation of JCFC NC. Importantly, highly stable codelivery of two drugs at a fixed 1:1 molar ratio in tumor cells in vitro further demonstrated higher apoptosis and enhanced synergistic anticancer activity compared to the individual free drugs and/or the mixture of CPT and FUDR. Celator Pharmaceutials, Inc. (previously; now a subsidiary of Jazz Pharmaceuticals) is a clinical-stage biopharmaceutical company for transforming the science of combination therapy and developing anticancer products.116 Celator’s proprietary technology platform enables the rational design and rapid evaluation of optimized combinations

Figure 3. Schematic route of FdU−BdM twin drug and construction of self-assembled nanoparticles for cancer therapy. Adapted with permission from ref 106. Copyright 2015 American Chemical Society.

MDR in tumor cells and therefore represent an excellent anticancer nanoplatform. Besides, this new method is quite reliable to self-deliver anticancer drugs. A very similar approach was utilized by Huang et al.44 to conjugate two drugs such as irinotecan and chlorambucil, having dissimilar solubilities. Irinotecan, a water-soluble anticancer drug, induced cytotoxicity directly by damaging DNA or inhibiting transcription in affected cells,109 while another water-insoluble drug, chlorambucil, worked as a DNA-alkylating agent to trigger cell death.110 The conjugated drug first dissolved in DMSO was further diluted in water to yield self-assembled amphiphilic NPs that could be easily internalized by tumor cells. The presence of an ester bond in the conjugated NPs was subsequently hydrolyzed after internalization resulting in the release of free drugs for excellent anticancer therapy and also overcoming MDR. Janus Drug−Drug Conjugate (JDDC). ADDC can easily self-assemble into nanodrugs; however, self-assembly behavior and final structure cannot be controlled as needed, which limits the clinical translation of ADDC. A variety of ADCs have been developed which demonstrate the ability to assemble into NPs with or without the presence of linker. In an example, Pramod et al. developed polysaccharide nanovesicles that can codeliver a hydrophilic and hydrophobic DOX and CPT, respectively, using a single polymer scaffold.111 However, such a nanoconstruct could only deliver varying ratios of anticancer drugs with a limited drug loading efficiency; for instance, in the presence of esterase enzyme 100% CPT and 65% DOX, while without esterase enzyme 55% and 35% of drugs, respectively, were released in vitro at 48 h. Although delivering amphiphilic drugs in a single nanoconstruct could be beneficial in cancer treatment, controlling the release behavior of two pharmacodynamically different molecular entities is technically challenging as observed from previous results. In another interesting report, Li et al. successfully prepared a dual-targeting delivery system from the mixture of HCPT−MTX and DSPE−HA− MTX conjugates.112 The core−shell−corona DSPE−HA− MTX NPs encapsulating two anticancer drug conjugates having different solubilities, HCPT−MTX, exhibited high drug entrapment efficiency (∼91.8%) together with pH/ esterase-controlled release behavior. Compared to the aforementioned DDS, this NP exhibited active targeting and synchronous dual-drug release at a nearly fixed drug-to-drug G


Review


To expand the scope of liposome-alike DDS and overcome associated disadvantages, Liang et al. recently envisioned an amphiphilic NCs from a highly symmetric Janus camptothecinfloxuridine conjugate (JCFC) (Figure 4).46 The liposomemimicking NC (JCFC) was synthesized by conjugating two hydrophilic and hydrophobic drugs FUDR and CPT, respectively, to a multivalent pentaerythritol via a hydrolyzable ester linkage.120 The as-synthesized amphiphilic JCFC NCs self-assembled in a 1:1 ratio of CPT/FUDR resulting in a uniform biphasic structure, also known as Janus (Figure 5). The in vitro and in vivo results depicted very high drug loading content, no premature release, long blood retention time, and enhanced tumor accumulation via EPR effect. The high loading and stability were due to the use of the drug−drug conjugate as a major ingredient in the formation of JCFC NC. Importantly, highly stable codelivery of two drugs at a fixed 1:1 molar ratio in tumor cells in vitro further demonstrated higher apoptosis and enhanced synergistic anticancer activity compared to the individual free drugs and/or the mixture of CPT and FUDR. JDDC Based Ternary Cocktail Chemotherapy to Overcome Resistance and Metastasis. JDDC-employed nanomedicines can be expanded further to combine multiple drugs or therapeutic agents, with temporally controlled drug release profile and simultaneously afford synergistic therapeutic efficacy against MDR, metastasis, and tumor recurrence. At present, triple-negative breast cancer (TNBC) is one of the most dangerous and refractory diseases prevalent in women worldwide and hardly responds to the standard treatment methods (hormonal or targeted therapies) except chemotherapy or surgical excision.121 Even after conventional treatment, TNBC still displays higher metastases and recurrence rate. According to a popular hypothesis about cancer stem-like cells (CSCs), it is proposed that rare residual CSCs are the major cause of tumor metastasis, recurrence, and the development of drug resistance.122 In addition, epithelialto-mesenchymal transition (EMT) is also profoundly active in CSCs generation and malignancy.123,124 Therefore, targeting all the identified sources that directly or indirectly contribute to the generation of CSCs and therefore augment malignancy could be a possible remedy. In this regard, Zhang et al. recently designed a ternary cocktail chemotherapy comprising Lovastatin as a major therapeutic ingredient that was successfully loaded in a previously reported CF NCs (LCF NCs).125 These NCs exhibited excellent drug loading capacity (68.3% for CF and 2.8% for L), high blood stability and circulation time, uniform size (∼107 nm), and sustained drug release performance (t1/2 (lov) = 307.8 min and t1/2 (CF) = 320.1 min) in vivo. Interestingly, the author compares the therapeutic activity of LCF NCs to a game “whack-a-mole” in which three drugs (also refers to three hammers) tactically hit TNBCs to prevent it from spreading. Benefited from the synergistic effect of these three drugs, LCF NCs have amplified the therapeutic effect by eradicating the tumors and CSCs. As shown in the results, the obtained LCF NCs were not only able to inhibit the growth of TNBCs both in vitro and in orthotopic tumorbearing animal models, but also suppressed pulmonary metastases (85.2% reduction compared to the control group), and no cancer recurrence was observed during 20 days post observation (Figure 6). Therefore, this paradigm provides a highly efficient but simple strategy for the development of simultaneous and synergistic triple chemotherapy. However, one of the major limitations is that the

Figure 4. Schematic illustration of the chemical structure of JCFC and its self-assembly into the liposome-like NC for cancer combination therapy. Adapted with permission from ref 46. Copyright 2018 John Wiley and Sons.

Figure 5. (a) TEM micrograph of JCFC NCs. In vitro (b) CPT and (c) FUDR release from JCFC NCs in PBS (pH 7.4) containing (or not) esterase (30 U/mL) and PBS (pH 5.0) containing (or not) esterase (30 U/mL) at 37 °C. Adapted with permission from ref 46. Copyright 2018 John Wiley and Sons.

incorporating traditional chemotherapies as well as molecularly targeted agents thought to enhance anticancer activity. Previously, the company had successfully conducted the clinical development of a nanoliposomal formulation comprising irinotecan and floxuridine (CPX-1). The CPX-1 formulation is a standard treatment regimen for metastatic colorectal cancer.117,118 The in vivo delivery of irinotecan and floxuridine (FUDR) coencapsulated in a liposome at a molar ratio of 1:1 (CPX-1) demonstrated enhanced therapeutic efficacy compared to both simple mixtures of two drugs in saline or individual liposomal agents.117,118 Nevertheless, liposome-based nanocarriers coencapsulating more than one drug have some major limitations, such as limited drug loading and encapsulation efficiency, stability, optimization of the drug ratio, and controlled release of chemically disparate drugs over time. Most importantly, fabrication of NPs having a perfect biphasic structure to deliver a fixed ratio of drugs (>1) is still a major challenge for scientists.119 H


Review


not only in nanoformation but also in the form of gas-filled phospholipid shell MBs. Such MBs offer limitless opportunities as a US contrast agent specially for the tissue delineation and perfusion imaging;131 moreover, they can also serve as a DDS to load various therapeutics.132 The latter one has been thoroughly achieved by either binding drugs to the MB shell directly or attaching drug loaded liposomal carriers to MB shells via site-specific ligands.133,134 However, higher drug loading in the context of MBs is a challenging task especially due to the compromised physical properties such as shell thickness and surface area.133,135,136 This can be possibly overcome to some extent by taking into account a special property of MBs known as UTMD.136−139 This technique utilizes the MBs inertial acoustic cavitation to create sonoporation after exposure of US at the targeted site. In contrast to the passive accumulation of NPs, the MBs-induced sonoporation can facilitate the accumulation of NPs in tumors beyond the EPR effect and largely accounts for improving the drug delivery and uptake by tumor cells. However, the limited drug loading capacity of MBs is still a major challenge and should be addressed by considering novel chemistries in the process of fabrication. Taking advantage of this, Liang et al. recently developed CF MBs based on the JCFC that could be self-assembled and filled with a gaseous core without necessarily adsorbing or conjugating drugs to the MBs surface (Figure 7).140 This newly adopted technique greatly solved the

Figure 6. (a) In vivo fluorescence of tumor regrowth 14 days after debulking surgery. (b) Representative photographs of metastatic tumors 30 days after surgery for each treatment group. Adapted with permission from ref 125. Copyright 2018 American Chemical Society.

tumor-bearing mouse model used as in vivo model does not fully recapitulate the clinical state of TNBC. In future, the novel LCF NC can be extended further to incorporate other therapeutic agents and use the more realistic animal model to treat TNBCs. This would consequently improve preclinical procedures and therapeutic outcomes for TNBC treatment in the future. JDDC Based Chemo-Photothermal Therapy to Overcome Drug Resistance and Recurrence. The nanostructure of CF NCs can be exploited further to load a variety of therapeutic and diagnostic agents, possibly inside the aqueous core or the bilayer membrane, and therefore serve as a powerful multimodal theranostic platform. Photothermal therapy (PTT), which employs near-infrared (NIR) light absorbers to convert light energy into heat, has attracted recent interest in chemo-photothermal therapy due to its minimal invasiveness and potential effectiveness.126,127 It is widely accepted that the photothermally induced hyperthermia can increase the cellular metabolism and membrane permeability.128 The advantage of combining photothermal and chemotherapy can therefore promote cellular uptake of drug and augment cellular cytotoxicity to a level which is nearly impossible to attain at lower drug doses. The chemotherapy can further inhibit the residual cancer cells after PTT, improving the synergistic treatment effect. In a recent example, Gao et al.129 proposed a new technique to incorporate a nearinfrared absorber, DiR, to enhance the therapeutic ability of CF NC. The amphiphilic CF together with DiR and PEGylated phospholipid (DSPE-PEG2000) self-assembled to form liposome-like structures in an aqueous solution. The stealth PEGylated corona helped NCs to prevent opsonization and accumulation in the RES organs such as liver and spleen.130 In addition, the obtained CF-DiR NCs have significantly high loading content, no premature release, stable codelivery of two drugs, and excellent photothermal conversion efficiency. A very high tumor accumulation of CF-DiR NCs after i.v. injection was clearly evident in the fluorescence imaging in vivo. Moreover, additional results showed that the CF-DiR NCs mediated chemo- and photothermal dual therapy was more cytotoxic to tumor cells than the chemotherapy or PTT alone. The absence of tumor recurrence during the entire treatment time further validated the synergism of the combination therapy. In brief, all results highlighted that CFDiR NCs could serve as a potential remedy against intrinsic resistance to chemotherapeutics via imaging-guided chemophotothermal therapy of cancer. JDDC MBs for Ultrasound Targeted Delivery. Among all the ADDCs, JDDC has shown great potential for clinical application due to its ability to self-assemble like phospholipids

Figure 7. Schematic illustration of engineering and ultrasonically induced micro-to-nanoconversion of Janus camptothecin-floxuridine microbubbles (CF MBs) for in situ combined cancer chemotherapy. Adapted with permission from ref 140. Copyright 2018 Springer Nature: Nature Asia Materials.

problem of limited drug loading in MBs. As compared to the conventional MBs having drug loading capacity usually

Amphiphilic Drug Conjugates as Nanomedicines for Combined

Recommend Documents