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Intracellular Peptide Self-Assembly: A Biomimetic Approach for in Situ Nano-drug Preparation Wei Du, Xiaomu Hu, Weichen Wei, and Gaolin Liang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00798 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018

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Bioconjugate Chemistry

Intracellular Peptide Self-Assembly: A Biomimetic Approach for in Situ Nano-drug Preparation Wei Du,† Xiaomu Hu,‡ Weichen Wei† and Gaolin Liang*,† †

CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China

‡

Department of Medicinal Chemistry, School of Pharmacy, The Fourth Military Medical University, Changle West Road 169, Xi'an, Shanxi 710032, China E-mail: [email protected]

ABSTRACT: Most of the nano-drugs are pre-prepared by encapsulating or loading the drugs with nano-carriers (e.g., dendrimers, liposomes, micelles, and polymeric nanoparticles). However, besides the low bioavailability and fast excretion of the nano-drugs in vivo, nano-carriers often exhibit in vitro and in vivo cytotoxicity, oxidative stress, and inflammation. Self-assembly is a ubiquitous process in biology where it plays important roles and underlies the formation of a wide variety of complex biological structures. Inspired by some cellular nanostructures (e.g., actin filaments, microtubules, vesicles, and micelles) in biological system which are formed via molecular self-assembly, in recent decades, scientists utilized self-assembly of oligomeric peptide under specific physiological or pathological environments to in situ construct nano-drugs for lesion-targeted therapies. On one hand, peptide-based nano-drugs always have some excellent intrinsic chemical (specificity, intrinsic bioactivity, biodegradability) and physical (small size, conformation) properties. On the other hand, stimuli-regulated intracellular self-assembly of nano-drugs is quite an efficient way to accumulate the drugs in lesion location and can realize an in situ slow release of the drugs. In 1

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this review article, we provided an overview on recent design principles for intracellular peptide self-assembly and illustrate how these principles have been applied for the in situ preparation of nano-drugs at the lesion location. In the last part, we listed some challenges underlying this strategy and their possible solutions. Moreover, we envisioned the future possible theranostic applications of this strategy.

KEYWORDS: peptide, intracellular self-assembly, nano-drug, therapy

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INTRODUCTION Theoretically, due to the specific properties of nanoscale particulate, a nano-drug system offers several pharmacokinetic advantages such as more specific drug delivery, higher metabolic stability and membrane permeability, improved bioavailability, longer circulation time in the blood than free drugs.1-4 To date, several nano-drug systems, including carrier-assistant drug delivery systems (DDSs) and drug self-delivery systems (DSDSs), have been developed to additionally improve their drug delivery efficacy and treatment effect, reduce their side effects, and overcome drug resistance of their pharmaceutical substances.5,6 Nevertheless, if the drug delivery systems are designed in nano forms (e.g., DDSs and DSDSs), their inherent nanotoxic potential and the shortcomings of the nano-scaled structures as listed below are hardly to overcome. First, depending on their physicochemical properties (e.g., chemical composition, size, surface modification), nano-drugs sometimes induce genetic damage, oxidative stress, even the inhibition and death to the cells.7-10 Second, DDSs always have low drug-loading capacity and inefficient drug release at the lesion site. Moreover, metabolites of the nano-carriers without direct therapeutic efficacy sometimes impose short-term or long-term toxicities on the living subjects.11 Third, the unintended uptake of the nanodrugs by the reticuloendothelial system (RES)-rich organs (e.g., liver and spleen), is usually inevitable.12 Self-assembly is a ubiquitous process and plays important roles in biology. It underlies the formation of a wide variety of complex biological structures.13 For example, nanostructures of cytoskeletons are resulted from self-assemblies of actins and tubulins and those obtained by self-assembly of DNA serve as storage for genetic information.14 Self-assembly occurs at all 3



scales.15 Molecular self-assembly is a ‘bottom-up’ fabrication process that molecules autonomously organized into ordered aggregates (spatial and/or temporal ordering) due to their mutual interactions via noncovalent forces.13 In recent years, peptide-based self-assembly has attracted increasing attentions due to their unique advantages such as convenient modification with functional motifs, easiness of synthesis and modular design, good biocompatibility and biodegradability, low immunogenicity and toxicity, and fast responses to various stimuli.16,17 There are a lot of methods to trigger and/or drive the peptide self-assembly normally by gradually changing the environmental conditions (e.g., pH, temperature, ionic strength, building block concentration, solvent, light, or enzyme activity).13,18,19 In recent decades, scientists have utilized the self-assembly of oligomeric peptide under specific physiological or pathological environments to in situ construct nano-drugs for lesion-targeted therapies. Note, these peptides (or prodrugs) are firstly administrated as small molecular forms but specifically concentrate at the pathological sites and convert to nano-drugs after in situ stimuli-triggered self-assembly. This in situ self-assembly has two obvious advantages as: first, peptide-based nano-drugs always have some excellent intrinsic chemical (specificity, intrinsic bioactivity, biodegradability) and physical (small size, conformation) properties; second, stimuli-regulated in situ self-assembly of nano-drugs is quite an efficient way to accumulate the drugs at lesion location, prevent diffusing through efflux pumps, and realize a slow release of the drugs. In this review article, we firstly discuss and summarize the designing principles for intracellular peptide self-assembly. Then, we focus on recent advances of in situ preparation of nano-drugs at the lesion locations. Finally, an outlook and perspective in this field of intracellular self-assembly of nano-drugs is discussed. 4


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DESIGNING PRINCIPLES OF THE PEPTIDE-BASED PRO-DRUGS FOR IN SITU NANO-DRUG PREPARATION Intracellular artificial nanostructures are resulted from different methods which should share the following common features: 1) the nanostructures are resulted from the self-assembly of building blocks at specific locations exclusively inside cells; 2) the self-assembly event is triggered by or coupled with an endogenous cellular process; and 3) the self-assembly endows the nanostructures with desired functions/effects at the specific locations.20,21 Compared with normal tissues, pathological ones such as tumors always exhibit vascular abnormalities (e.g., weak acidity (~ pH 6.8), abnormal temperatures, over-expressed proteins and enzymes, hypoxia, high levels of metabolites, reactive small molecule species, etc). Moreover, upon cellular uptake, prodrugs are subjected to intracellular pH gradients (pH 5.9–6.2 in early endosomes and pH 5.0– 5.5 in late endosomes and lysosomes), redox gradients, and H2O2 gradients within different cell organelles or the cytosol.22 By carefully designing, these features could be employed to trigger the intracellular self-assembly of peptide-based prodrugs into nano-drugs specifically at pathological sites and slow release of therapeutic components thereafter. While at the normal tissues, the small-molecular prodrugs are more likely to be cleared out through urinary excretion, a process that occurs only when the size of the agent is smaller than the physiologic pore size of filtration slit in the glomerular capillary wall (~ 5 nm).23 In principle, self-assembly requires a concentration over the critical micelle concentration (CMC) and a suitable amphiphilicity of the build block. Thus, the general strategy is firstly by conjugation of a response segment to the peptide to generate a non-self-assembling hydrophilic precursor that specifically targets the pathological site. On one hand, the targeting 5



process can enrich the self-assembling molecules at the pathological site to promote the self-assembly process. On the other hand, at the pathological site, the response segment is able to convert the hydrophilic precursor to the amphiphilic self-assembling monomer via stimuli-controlled bond cleavage or the formation of bigger aromatic ring. This conversion generates strong noncovalent forces (e.g., hydrophobic interactions, π-π stacking, hydrogen bonding, or van der Waals forces) to initiate self-assembling of monomers into nano-drugs in nanofibers or nanoparticles. Then, the therapeutic components are slowly released in situ in response to the environmental cues (Figure 1). Herein, the self-assembling monomer can be either pure peptide or peptide derivatives. According to the function of the peptide and the conjugation method between the peptide and the drug, there are three general approaches to in situ self-assemble nano-drugs. 1) Intracellular self-assembly of innoxious peptide to form nanostructures as nano-drugs to induce cell death (Figure 1a). 2) Intracellular co-assembly of the peptide with drug monomers. In this case, peptide serves as the self-assembling motif and the physically mixed drugs co-assemble with the peptide to afford nano-drug (i in Figure 1b); or both the peptide and the prodrugs response to the stimuli to co-assemble into nano-drugs (ii in Figure 1b). 3) Intracellular self-assembly of drug-peptide conjugates. The peptide is covalently conjugated with the drug and, upon stimuli regulation, the drug-peptide conjugate self-assembles into nano-drug (Figure 1d). In the following sections, we summarize the recent advances of in situ preparation of nano-drugs following above classification.

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Figure 1. Schematiic illustratio on of the dessign principlles of in situ u preparation on of nano-d drugs. (a) Intracellular self-asssembly of innoxious i peeptide to form nanostru uctures as naano-drugs to o induce cell deaath. (b) Intraacellular co-assembly off the peptide with drug monomers ((i) with pu ure drug, (ii) withh prodrug). (c) ( Intracellu ular self-asssembly of drrug-peptide conjugates. RECEN NT ADVAN NCES OF IN I SITU NA ANO-DRUG G PREPAR RATION

Intraceellular self-aassembly of innoxiouss peptide to o form nan nostructuress as nano-d drugs to induce cell death Uninttended or unregulated u self-assemb mbly could be b detrimen ntal to norm mal life acttivities,16 which hhas been evvidenced by y some neur urodegenerattive diseases caused byy the misfo olding of amyloidd proteins.244 However, when this process haappens at th he pathologgical sites, it might surprisinngly be a treeatment of the t diseases.. For examp ple, Svanborrg et al. repoorted that thee protein aggregaates formed by the selff-assembly oof partially unfolded u α--lactalbuminn (HAMLET T) could induce apoptosis of tumor cells.25 Siince intraceellular selff-assembly requires a higher CMC, it is important to t spatiotem mporally con ntrol the concenttration of thhe monomer than its C 7



enrichment of the monomers for the self-assembly. M.T. Jeena et al reported a specific cellular organelle localization-induced supramolecular self-assembly (OLISA) system in which self-assembly was induced by raising local concentrations of the self-assembling molecules without extra treatment.26 Since disruption of mitochondria membrane and subsequent release of the contents (e.g., cytochrome c) from mitochondria is a major event in the intrinsic cell death signaling pathway,27,28 targeting mitochondria-targeting induced cancer cell death29,30 (e.g., modulating the redox potential of mitochondria31) may be advantageous in overcoming the multidrug resistance (MDR) in cancer therapy.29 They designed and synthesized a mitochondria-targeting peptide amphiphile (1) which was equipped with a triphenyl phosphinium (TPP) group for mitochondria-targeting and a pyrene group for the fluorescence imaging of mitochondria after self-assembly (Figure 2a). With the increase of the mitochondrial membrane potential of cancer cells, 1 was accumulated in the mitochondrial, leading to the self-assembly to form the fibrious nanostructures. The intra mitochondrial fibrils further disrupted the mitochondria membrane resulting in the leakage of mitochondrial contents to the cytosol, which eventually induced cellular apoptosis (Figure 2b). Further examination of mitochondrial co-localization of 1 with MitoTracker Red FM showed high localization of 1 inside mitochondria (Figure 2c), indicating the excellent mitochondria-targeting ability of 1. Since both cellular membrane and mitochondrial membrane of cancer cells show much higher negative potential than those of normal cells,32 their experimental results indicated that the accumulation of 1 inside normal cell lines was approximately 10-fold less than that in cancer cell lines. As a result, 1 showed much higher toxicity towards cancer cell lines than normal cell lines. The control peptide 1G which just formed spherical assembly (data not shown here) 8


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showedd less cell innhibition th han other m molecules (i..e. 1, 1V, 1Fx) 1 whichh formed naanofibers inside ccells (Figurre 2d). Furth her cell staiining and fllow cytometry analysess indicated an early apoptossis mechaniism for 1 to induce ccell death (Figure 2ee). Thereforre, this straategy of organellle-targeted accumulatio a on and in siitu self-assem mbly of a peptide p ampphiphile cou uld be an efficient nt way to conntrol cellularr fate.

Figure 2. Intra-miitochondriall self-assem mbly for can ncer therapy y. (a) Strucctural design n of the 9



mitochondria-targeting peptide amphiphile (1), and the control peptides (1V, 1Fx, 1G). (b) Illustration of the intra-mitochondrial self-assembly of 1. (c) Mitochondrial co-localization of 1 measured with MitoTracker Red FM shows high localization inside mitochondria (scale bar, 5 μm). (d) Toxicity analysis of 1, 1Fx, 1V and 1G in different cancer cell lines (i.e., HeLa, MDA-MB-468, PC3 and SCC7 cell lines) and normal cell lines (i.e. HEK 293T and NIH 3T3 cell lines). Data represent mean ± s.d. from three independent experiments. (e) Annexin V/PI assay and flow cytometric analysis of cell death induced by 1, which showed early apoptosis (scale bar, 10 μm). Adapted with permission from ref. 26. Copyright 2017. Nature Publishing Group. Xu group raised the concept of enzyme-instructed self-assembly (EISA) as a promising approach for cancer therapy.14 In fact, using apoptosis as an example, the combination of enzymatic transformation and self-assembly, as a multistep process, constitutes an inherent feature of apoptosis (Figure 3a).33 Intracellular EISA can interact with multiple proteins to interrupt multiple proliferation process, interfere with redundant pathways, and elude efflux pump. Cell surface EISA can block the cellular mass exchange, prosurvival signals, and metastasis. By careful design of the precursors which target the enzymes highly expressed in cancer cells, the EISA process could be a potential strategy for developing anti-cancer therapeutics that selectively kill cancer cells but not normal cells. Diphenylalanine (FF) peptide, which was frequently used by Xu et al as the core recognition motif for molecular self-assembly, was extracted from Alzheimer’s β-amyloid polypeptide.34 FF-based assemblies have shown remarkable advantages including functional versatility, ease of production, biodegradability, biocompatibility, and non-immunogenicity. By combining the FF group with an enzyme-responsive hydrophilic motif, Xu and coworkers reported a series of FF-based peptides which subject to intracellular EISA processes to induce 10


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cell death. As exampled in Figure 3b, a FF peptide precursor 2 containing an enzyme (i.e., esterase)-cleavable bond (i.e., an ester bond), was subjected to esterase-cleavage to yield the amphiphilic product 2’ which self-assembled into nanofibers.20 When 2 was applied to incubate with esterase-overexpressing cancer cells, intracellular EISA process of 2 could induce the cancer cell death. Unlike the intracellular esterase, some enzymes (e.g., matrix metalloproteinase-7, MMP-7) are excessively expressed by cancer cells but secreted and activated excellularly.35 Maruyama et al. recently reported an innovative approach that used MMP-7 to convert the precursor 3 into 3’ which translocated the cancer cell membranes and self-assembled into nanofibers, critically impairing the cellular functions and inducing cancer cell death in the end.36 Another important enzyme, alkaline phosphatase (ALP), responds for the dephosphorylation of some small molecules (or macromolecules) which are usually building blocks of DNA or proteins.37 ALP activity usually increases in several human diseases, including bone diseases (Paget's disease, osteomalacia, osteoblastic bone cancer) and liver diseases (hepatitis, obstructive jaundice, cancer).38 Moreover, ALP was found highly expressed on (and out of) the cell membrane of some cancer cells (e.g., HeLa, MES-SA, and MES-SA/Dx5 cells).39 Therefore, ALP-instructed self-assembly processes can be used to control cell fates by either interrupting the intracellular multistep processes or blocking the mass exchange and communication among the cells (Figure 3a). As exampled in Figure 3b, the FF-based, EISA precursors in L-form or D-form (i.e., 4 or 5) was designed for ALP-dephosphorylation to yield the enzymatic products 4’ or 5’ which self-assembled into nanofibers with different cancer cell-killing abilities.40-42

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Figure 3. (a) Plauusible mechanism of EISA to inhibit i canccer cells: ((I) interacting with t interruptt multiple pproliferation n process, (II) ( interferiing with reedundant multiplee proteins to pathwayys, (III) eluuding efflux x pump, andd (IV) blocking cellulaar mass excchange, pro osurvival signals,, and metastasis. Adapted from reef. 33. Copy yright 2014. Americann Chemical Society. (b) Som me representtative peptid de-based prrecursors ussed for EISA A-mediated cancer therrapy. As w we know, sm mall differeence of the monomerss might resu ult in huge difference in their EISA aability. Theerefore, it iss importantt to screen the monom mers with ddifferent molecular m featuress (or thermoodynamic properties) p tto increase the EISA efficacy for better inhib bition of cancer ccells. Feng et al design ned and synnthesized a series of sttructural annalogues of peptidic precursors (6, 7, 8, 8 9, 10 an nd 11 in Fiigure 4a) and a screeneed their canncer cell-in nhibitory

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abilities.44 These precursors have the same peptidic backbone (i.e., FF) and an ALP cleavage site but differ in N-terminal capping, C-terminal modification, stereochemistry, or regiochemistry. The authors measured the CMCs of the above precursors (6−11) and their corresponding dephophorylated products (6’−11’) and the results indicate that the CMC values of the precursors follow the order of 6 < 8 < 7 < 9 < 10 < 11. The self-assembling abilities of the dephosphorylated molecules congruously showed 6’ > 8’ > 7’ > 9’ > 10’ > 11’ (Figure 4b). Cytotoxicity results of these precursors towards ALP-overexpressing osteosarcoma cell line suggested that, it was the self-assembling ability but not the stereochemistry or the regiochemistry of the EISA precursors that determined their cancer cell inhibition ability (Figure 4c). The authors then examined the cytotoxicity of 6 and 10 (i.e., with the highest and much lower self-assembling ability) on three cell lines with different ALP expressions (breast adenocarcinoma cells MCF-7, glioblastoma cells T98G, and a normal stromal cell line HS-5).45 They found that the precursors consistently showed lower half-inhibitory (IC50) values on cell lines with higher ALP expression levels. Thus, the authors concluded that one parameter of the precursor’s self-assembling ability determines the thermodynamic properties of an EISA Process, while another parameter of enzyme expression level of cancer cells kinetically controls the EISA process. These two parameters can guide the design of the precursors for EISA-mediated anti-cancer therapy.

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Figure 4. (a) Moleecular Structtures of the Precursors (6 – 11). (b b) CMC vallues of preccursors 6 − 11 annd their corrresponding dephosphoorylated pep ptide derivaatives 6’ – 111’. (c) Corrrelation betweenn the self-asssembling ability a (−ΔG G0) and anticcancer activ vity (pIC50) of EISA molecules m against Saos-2 celll. (d) IC50 (72 h) of 6 oor 10 again nst Saos-2 cells, MCF-77 cells, T98 8G cells, or HS-55 cells. Adaapted from ref. r 44. Copyyright 2014 4. American n Chemical SSociety. Intraceellular co-asssembly of the t peptidee with drug monomers Althoough the inntracellular (or on celll surface) self-assemb bly of innoccuous peptide is a promising strategyy for cancerr therapy, thhe IC50 valu ues of the self-assembbling monom mers are always higher thaan 100 μM M.45,46 Morreover, only y those ceell-penetratinng monom mers and enzymee-overexpresssing cancerr cells coulld be applied to this system. Nev evertheless, a lot of efforts have still been b made to improvee the theraapeutic capaabilities of this approaach. For examplee, mitochonndria-targeting and moddulating its redox poteential thereaafter is a prromising approacch to kill cancer c cells with miniimum drug resistance. Employingg this, Wan ng et al developped a multi-ttargeting EIISA precursor (NBD-FF FYpK(TPP))) for cancerr cell inhibition.47 In this preecursor, a triiphenyl pho osphinium ((TPP) motiff was design ned for mitoochondria-taargeting, 14


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FFYp was used for ALP-triggered self-assembly to kill cancer cells, and an environmental sensitive NBD fluorophore was designed for fluorescence monitoring the self-assembly process. Their results indicated that the integration of EISA and subcellular targeting processes could synergistically killing cancer cells without acquired drug resistance. Wu et al introduced fluorine substitution to the precursors to enhance their self-assembling ability as well as anti-cancer capability.48 However, the therapeutic effect of these agents are still incomparable to the commonly used anti-cancer drugs. Doxorubicin and taxol always show IC50 values less than 10 μM towards various cancer cell lines.49-51 However, these chemotherapeutic agents suffer from several drawbacks including insufficient drug concentrations in tumors, systemic toxicity, lack of selectivity for tumor cells over normal cells, and the appearance of drug-resistant tumor cells.52,53 As we know, hierarchically ordered co-assembly of macromolecules with supramolecular building blocks (e.g., peptides, proteins, lipids, nucleic acids, and saccharides) inside cells is a fundamental organizational principle of living subjects. Co-assembly of two or more components provides opportunity to control the morphological, physical and mechanical properties of the co-assembled material that might not be found in the individual components.54 Liu et al verified that co-assembly of 10-Hydroxycamptothecin (HCPT) with peptide NapGDFDFDYGRGD could boost the selectivity and antitumor efficacy of free HCPT.55 When the co-assembly process was precisely controlled to happen at the pathological sites, some pharmacological advantages that the free drug does not have could be achieved. These advantages include avoiding the undesired uptake of the drug by RES-rich organs, increasing the cellular accumulation and blood circulation time of the drug, prolonging the therapeutic effect of the drug by slow release of the drug over an extended duration (days or months), and 15



overcoming the MDR of the tumors.56,57 Huang et al developed an in situ tumor-specific formation of enzyme-instructed supramolecular co-assemblies to enrich indocyanine green (ICG) for fluorescence/photoacoustic imaging-guided photothermal therapy (PTT).58 As shown in Figure 5a, precursor 12 was subjected to ALP-dephosphorylation to yield 13 which self-assembled in water to form nanofibers 14. For in vivo treatment, the initial mixing of ICG with 12 led to a micelle structure which gradually accumulated in the tumor site by enhanced permeability and retention (EPR) effect. Upon the catalysis of ALP, 12 was converted to 13 which co-assembled with ICG to form the ICG-doped nanofibers 15 in the tumor site. Within the nanofiber 15, ICG took the J-aggregate arrangement evidenced by its red-shifted and significantly enhanced absorbance. Co-assembled ICG showed partially self-quenched fluorescence and enhanced photoacoustic and photothermal signals. However, without co-assembly, ICG is excreted shortly via efflux pump. In situ formation of the ICG-doped nanofibers efficiently prolonged the retention time of ICG compared with free ICG, evidenced by the in vivo NIR fluorescence imaging of the tumor-bearing mice (Figure 5b). Upon laser irradiation, the ICG-doped nanofiber 15 could efficiently convert photons into tumor-damaging heat and effectively ablate the tumors (Figure 5c&d), while those tumors in control groups were slightly inhibited and grew at a much higher speed (Figure 5e). Based on the fact that the input photons were converted by ICG to either fluorescence emission or thermal energy, in this case, the enhanced absorption and decreased fluorescence of ICG promised it with better photothermal conversion efficiency. Thus, this in situ tumor-specific, enzyme-instructed co-assembly of ICG-nanofiber could be an alternative strategy for the PTT of cancers. 16


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It has been estimated that up to 40% of the chemotherapeutic drugs are hydrophobic and suffer from low solubility in blood and low bioavailability which may lead to rapid blood clearance and reduced chemotherapeutic efficacy.59 To resolve this problem, some hydrophobic drugs are modified with polar functionalities (e.g., sulfoxidation, phosphorylation, protonation of amine group, etc) to yield their hydrophilic (or amphiphilic) prodrugs.60 After these crafty modifications, some prodrugs are “smartly” responsive to the pathological conditions and converted to their active parent drugs. For example, ALP can catalyze the dephosphorylation of fosphenytoin and by this means to spatially control the release of the active drug phenytoin at the lesion site highly expressing ALP.61 Combination of such stimuli-responsive prodrug with stimuli-activatable peptide precursor could realize a co-assembly of the two components at the targeting site, resulting in a concentrating and thereafter slow release of the active drug. Tang et al confirmed that this “smart” strategy was promising to boost the therapeutic effect of a drug.62 As illustrated in Figure 5f, under the catalysis of the same enzyme ALP, the hydrogelator precursor Nap-FFYp (16) yielded the hydrogelator Nap-FFY (16’) while the water-soluble prodrug Dexamethasone sodium phosphate (Dp) yielded the anti-inflammation drug Dexamethasone (Dex). In the cytosol of ALP-overexpressing inflammatory macrophages, these two dephosphorylation products 16’ and Dex co-assembled to form Nanofiber 2 and the drug Dex was slowly released. Released Dex integrated with GR to form the Dex-GR complex which competitively bond with the transcriptional factor NF-kB elements (e.g., p65 and p50), and thus down-regulated the expression of the inflammation molecules. Cell experiments indicated that, at a concentration beyond the CMC, intracellular ALP-instructed co-assembly of Dp with 16 extensively boosted the anti-inflammation efficacy of Dex. As shown in Figure 5g, at Dp 17



concenttration of 25 μM, expression e of the infflammation moleculess (e.g., thee major histocom mpatibility complex c II (MHCII), ( C CD86/CD80,, and CD11cc in this worrk) in the ceells of 16 + Dp grroup was siggnificantly down-regula d ated compaared with thee control gro roups. Besid des small molecullar chemothherapeutic drugs, d macrromolecular drugs such h as vaccinnes are also o able to co-assem mble with peptides fo or enhancedd therapeuticc effect. Liiu et al repported a meethod of ALP-instructed, in situ preparration of peeptide-HIV DNA D vaccine co-assem mblies in which w the peptidicc nanofiber acted as the adjuvant tto improve the immun ne responsess of vaccinee against HIV.63

Figure 5. Intracelluular co-asseembly enhannces the theerapeutic efffect of a druug. (a) Princciple for enzymee-triggered supramolec s ular co-asseembly of IC CG and 12. (b) ( Represeentative in vivo v NIR fluoresccence imagees of 15 (IC CG, 10 mg/kkg; 12, 100 mg/kg, n = 4) and ICG G (ICG, 10 mg/kg, m n = 4) onn HeLa-tum mor-bearing mice after iintravenouss administraation. (c) Phhotothermall images of HeLa tumor-bearing mice at 24 and 448 h post in njection (IC CG at 5 mgg/kg, and 12 2 at 100 mg/kg) with exposure to 808 8 nm laser irradiation (0.8 W/cm m2, 5 min). ((d) Photogrraphs of 18


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HeLa-tumor-bearing mice at different days after PTT treatment (15 + laser). (e) Growth curves of different groups of HeLa-tumor-bearing mice after PTT treatment. Tumor volumes have been normalized to their initial sizes. Error bars represent the standard deviations of five mice per group. ***P < 0.001. Adapted from ref. 58. Copyright 2015. American Chemical Society. (f) Schematic illustration of ALP-instructed co-assembly of 16 and Dp to form Nanofiber 2 in vitro and in inflammatory macrophages. (g) Summary graphs of the relative amounts of inflammation molecules MHC-II, CD86, CD80, and CD11c in inflammatory BMDMs incubated with the compounds at 25 μM and 37 °C for 24 h. PC, positive control. The results are illustrated as the mean ± standard error of mean (SEM) of the relative fluorescence intensity of F4/80+ CD11b+ MHC-II + cells, F4/80 + CD11b + CD86+ cells, F4/80+ CD11b+ CD8o+ cells, and F4/80+ CD11b + CD11c + cells, respectively. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. Adapted from ref. 62. Copyright 2017. The Royal Society of Chemistry. Intracellular self-assembly of drug-peptide conjugates Despite the advances of abovementioned co-assembly strategy, it still has many unmet challenges and the biggest one is the spatiotemporal control of the co-assembly. For example, since the blood circulation time and the targeting-specificity of the co-assembling components differ from each other, it is hard for them to reach their critical aggregation concentrations (CACs) at the same location or simultaneously. Therefore, direct self-assembly of the peptide-based prodrugs or drug-peptide conjugates might overcome above challenges and has received considerable research attentions in recent years.64 Many drugs (e.g., olsalazine,65 camptothecin,66 taxol,67 cisplatin,68 etc) are able to be covalently conjugated to oligopeptides to yield amphiphiles with self-assembling abilities. With careful design of a drug-peptide conjugate, it is promising to realize the stimuli-instructed self-assembly of the conjugate and subsequent slow release of the drug at the lesion site. The 19



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abovementioned EISA strategy can be a good candidate to realize the intracellular stimuli-instructed self-assembly of drug-peptide conjugates. Besides intracellular enzymes, many bioactive molecules such as glutathione (GSH) can also trigger the intracellular self-assembly of drug-peptide conjugates to form the nanodrugs in situ. A series of drug-peptide conjugates which subject to stimuli-instructed intracellular self-assembly for cancer therapy have been developed and are listed in Table 1. Table1. Drug-peptide conjugates for intracellular hydrogelation Peptide

therapeutic

Stimuli

Application

ALP

drug delivery &

agents Nap-FFK(Taxol)Yp

Taxol

cancer therapy 69 Nap-DFDFDK(Taxol)DYp

Taxol

ALP

long term drug delivery & cancer therapy70

Taxol-DFDYp & Taxol-LFLYp

Taxol

ALP

cancer therapy71

P18-PLGVRGRGD

Purpurin 18

gelatinase

PA imaging and thermaltherapy of tumor72

Dex-FFFK(Taxol/HCPT)E-ss-E

Drug 1: Dex

E

Drug2:

GSH

co-delivery of drug & cancer therapy73

Taxol/HCPT 20


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Taxol-K(Ac)E-ss-EE

Taxol

GSH

Sustained drug release & cancer therapy74

Besides enzymes and bioactive molecules, some external stimuli such as pH and ionic strength are also able to trigger the self-assembly of drug-peptide conjugates. Tang et al rationally designed two hydrogelators (a pamidronate (Pami)-derivative Fmoc-FF-Pamidronate (17) and an alendronate (Alen)-derivative Fmoc-FF-Alendronate (18)) which could self-assemble into nanofibers to form supramolecular hydrogels under acidic conditions (Figure 6a).64 The rationale is that, after being administered in vivo, the hydrogelators will target and induce the apoptosis of osteoclasts by binding to the bone mineral and “smartly” self-assemble into nanofibers to locally concentrate the drugs at the acidic bone resorption Lacunae (Figure 6b). They investigated the osteoclastgenesis-inhibitory effect of compound 17, 18, Pami, and Alen on bone-marrow-derived monocytes/macrophage (BMM) cells. As shown in Figure 6c, at low concentrations that all the four compounds did not impose cytotoxicity to the BMM cells, neither Pami nor Alen showed inhibitory effects on the osteoclastogenesis of the BMM cells but 17 and 18 did. In vitro bone resorption assay indicated that, large bone resorption pits were observed in the control group while smaller and fewer resorption pits were observed in both 17 and 18-treated groups (Figure 6d). In comparison, one order of magnitude concentration of Pami or Alen was needed to achieve a comparable inhibitory effect on osteoclastic bone resorption to that of 17 or 18 (Figure 6e), suggesting that this self-assembling method of drug-peptide conjugates is a promising strategy to improve drug efficacy.

21



Figure 6. (a) Chem mical structtures of 17, Pami, 18, and Alen. (b) ( Schemattic illustration of in vitro suupramolecullar hydrogell formationn of 17 or 18 1 under accidic condittions, and plausible p mechannism of the bone-resorrption inhibiitory effectt of self-asssembled 17 or 18 at the t bone resorption lacunae. (c) The inh hibitory effeect of 17 and d 18 on osteeoclastogeneesis of BMM Ms at the indicateed concentrrations afterr 96 h inccubation. (d d) Represen ntative SEM M images of bone resorption after being treated with 17, 188, Pami, orr Alen at th he indicatedd concentrations. (e) Quantitaative analyssis of bone resorption arrea after bein ng treated with w 17, 18, PPami, or Alen at the indicateed concentraations.*, p < 0.05 vs. eaach control. Adapted fro om ref.75. C Copyright 2017 The Royal S Society of Chhemistry. Ligannd–receptor interactions can also ffacilitate thee self-assem mbly processs. Recently y, Tiller's group reeported an interesting i “surface “ indduced hydro ogelation” ph henomenonn that a hydrrogelator started tto gel on a surface s at a concentratioon 50 times below its minimum m geelation conceentration (MGC)..76 By emplloying the “surface-ind “ duced self-aassembly” strategy, Renn et al deveeloped a bacteriaal surface-innduced self-assembly sttrategy in which, w via th he specific interaction between

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the gram positive bacterial cell wall peptides and vancomycin (Van), the self-assemblies of two environment-sensitive fluorescent NBD–Van conjugates (NBD–FFYEGK(Van) and NBD– FFYEEGK(Van)) were initiated for simultaneous bacterial detection and inhibition.77 The abovementioned self-assembly strategies all used built-in self-assembly motifs (i.e., the self-assembling motifs were pre-prepared before being delivered on (or into) the cells). Certainly, it is feasible to in situ form small molecular self-assembly moieties. That is, pre-prepared small molecules do not contain self-assembling moieties, but at pathological site, stimuli trigger a chemical reaction on the molecules to yield the self-assembling motifs which self-assemble into nanostructures in situ. Rao and co-workers developed a smart strategy of intracellular self-assembly to in situ prepare nanostructures inside cells using a click condensation reaction between 2-cyanobenzothiazole (CBT) and D-cysteine (D-Cys).78 As exampled in Figure 7a, a general chemical structure of 19 is subjected to cellular stimuli (e.g., pH change, reduction, or enzyme) to yield the active intermediate 20. Then the click condensation reaction happens among the molecules of 20 to yield the macrocyclized dimer 21 or oligomers 22 which self-assemble into nanostructures. Employing this click condensation reaction, Yuan et al reported a taxol derivative Ac-RVRRC-(StBu)-K(taxol)-CBT (CBT-Taxol, 23, Figure 7b) which was subjected to intracellular furin-controlled condensation and self-assembly of taxol nanoparticles (Taxol-NPs) to overcome the MDR of tumor cells.79 The mechanism is illustrated in Figure 7c. In detail, after entering furin-overexpressing cells (HCT 116 cells in this work), 23 was subjected to GSH-reduction and furin-controlled cleavage to expose the thiol group and amine group of its cysteine motif. Then the condensation reaction happened to yield the 23



hydrophobic oligomers (mostly dimers)

which self-assembled into taxol nanoparticles

(Taxol-NPs) at (or near) the locations of activated furin (i.e. Golgi bodies), as demonstrated previously.80,81 As-formed Taxol-NPs were difficult to pump out by P-glycoprotein and, therefore, their circulation time inside cells was effectively prolonged. After the ester bonds on Taxol-NPs were cleaved by the esterases in cells, free taxol was gradually released to bind the tubulin which effectively overcame the MDR of the cancer cells. Cytotoxicity studies indicated that CBT-Taxol 23 had a 4.5-fold increased anti-MDR effect on taxol-resistant HCT 116 cancer cells than free taxol (Figure 7d). In vivo results indicated that, 23-treated mice showed impressively better antitumor effects than those injected with taxol on both HCT 116 tumors and taxol-resistant HCT 116 tumors (Figure 7e). At day 20, 23 showed in vivo anti-MDR factor 1.53-fold of that of taxol to the tumors (Figure 7f). Body weight curves of the tumor-bearing mice during treatment suggested that, while 23 had better anti-MDR effect than taxol, it also remitted the toxicity of taxol to the mice (Figure 7f). This study provided an alternative strategy of in situ nano-drug preparation overcome tumor MDR with enhanced efficiency.

24


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Figure 7. (a) Propoosed two-sttep condenssation of mo onomers, co ontrolled by pH, reduction, or a proteasee. b) Cheemical stru uctures off 23. c) Schematic illustrationn of intraacellular furin-coontrolled seelf-assembly of Taxool-NPs for anti-MDR.. (d) Cell viability study s of parental HCT 1166 cells and taxol-resisttant HCT 116 cells on o treatmennt with taxo ol or 23 (co-incuubated withh 50 μM Ac-RVRRCA -(StBu)-K(ttaxol)-CBT,, 24) for 488 h. (e) An ntitumor effect oof control vehicle, v taxol, and 23 coinjected with 24 on n parental H HCT 116 tu umor or taxol-reesistant HCT T 116 tumo or implantedd in nude mice. m (f) Tum mor weight at day 20 and a body weight of nude miice implantted with paarental HCT T 116 tumors or taxol--resistant HCT H 116 tumors after beingg treated wiith control vehicle, tax xol, and 23 3 coinjectedd with 24. Adapted A C KGaA, W Weinheim. from ref. 79. Copyyright 2015. WILEY-V CH Verlag GmbH & Co. SUMM MARY AND D OUTLOO OK In reecent years, great strid des have beeen made in i the investigation off in situ naano-drug preparattion throughh intracellullar peptide self-assemb bly strategy. By selectiing suitable triggers (e.g., pH H, bioactivee molecules, or enzymess) that relatee to the diseases, it is feeasible to co ontrol the 25



self-assembly of the peptide precursors (or drug-peptide conjugates) on the pericellular (or intracellular, or subcellular) space to form nanodrugs to enhance the therapeutic effect of the prodrugs/precursors. This trigger-controlled intracellular self-assembly only occurs and elicits its desired functions/effects at the specific locations where the trigger of interest is particular rich. This feature improves the selectivity of a drug, helps reducing its side effects and overcoming the drug-resistant of a disease, which is superior to the direct use of the drug, drug assemblies, or drug aggregates. By replacing the drug with other functional moieties (e.g., an imaging probe) in the precursors, it is promising to evolve the intracellular self-assembly strategy for molecular imaging,82-84 signaling transduction,85 etc. However, the development of in situ peptide-based nano-drug is still in its infancy, there remain many issues and challenges to be resolved. 1) Small peptide is always susceptible to proteolytic degradation by endogenous enzymes, such as peptidases or amidases, which endangers the stability of prodrugs/precursors and assemblies in physiological environment. 2) It is hard to obtain the morphologies and the structures of the assemblies, and the mechanisms of some assemblies to regulate cell behaviors remain unknown.21 3) Disease treatment is closely tied to its diagnosis, however, early diagnosis of some diseases (e.g., cancer) is irrealizable to date. Thus, it is more important to develop efficient early diagnostic approaches before applying this strategy for therapy. 4) Compared with as-prepared nano-platforms, small molecular precursors/prodrugs lack interspaces to realize an all-in-one system (e.g., multi-modality). Intentional enabling the precursors/prodrugs with multi-functionality will more likely increase their complexity, as well as change their physicochemical properties (e.g., water solubility).

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In the last years, scientists have spared no efforts to meet these challenges. For examples, several labs have improved the stability of the peptides towards proteolysis by replacing the L-amino acids with D-amino acids.16 Glycosylation is another promising strategy to enhance the in vivo stability of the peptides since nature already used this strategy, as demonstrated by glycosylated RNase B.86 The rapid development of advanced technologies such as cryo-electron microscopy (cryo-EM) will benefit the scientists to explore the morphologies and the structures of the assemblies, as well as their mechanisms to regulate cell behaviors. Great efforts have been made to the early diagnosis of cancer and numerous achievements have been obtained at the experimental state.87 For example, developments of theranostic agents wherein the therapeutic drugs are combined with diagnostic probes will increase the insights into the delivery kinetics, local response, and overall efficacy of the drugs.88 By simultaneously immobilizing the therapeutic agent and imaging moieties into the self-assembling precursors, we anticipate to construct a “smart” intracellular theranostic platform for more efficient disease diagnosis and therapy in the near future. We envision herein that, with the development of new technology and methodology, this strategy of in situ nano-drug preparation through intracellular peptide self-assembly go beyond the conceivable achievements in the future.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

27



ACKNOWLEDGEMENTS This work was supported by Collaborative Innovation Center of Suzhou Nano Science and Technology, the Major program of Development Foundation of Hefei Center for Physical Science and Technology (2016FXZY006), Ministry of Science and Technology of China (2016YFA0400904), and the National Natural Science Foundation of China (Grants 21725505 and 21675145).

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