Peptide dendrimer-Doxorubicin conjugate-based nanoparticles as an enzyme-responsive drug delivery system for cancer therapy.

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Peptide Dendrimer–Doxorubicin Conjugate-Based Nanoparticle as an Enzyme-Responsive Drug Delivery System for Cancer Therapy Chengyuan Zhang, Dayi Pan, Kui Luo,* Wenchuan She, Chunhua Guo, Yang Yang, and Zhongwei Gu* including lipids (liposomes),[6] polymeric nanoparticles,[7,8] micelles,[9] and denPeptide dendrimers have shown promise as an attractive platform for drug drimers, have been designed for cancer delivery. In this study, mPEGylated peptide dendrimer–doxorubicin (dentherapeutic applications, advancements drimer–DOX) conjugate-based nanoparticle is prepared and characterized of novel drug delivery systems, which as an enzyme-responsive drug delivery vehicle. The drug DOX is conjugated can maximize the efficiency of drugs to the periphery of dendrimer via an enzyme-responsive tetra-peptide linker while minimizing drug-related toxicities, Gly-Phe-Leu-Gly (GFLG). The dendrimer–DOX conjugate can self-assemble remains challenge.[10] Dendrimers are particularly suitable into nanoparticle, which is confirmed by dynamic light scattering, scanning candidates for the delivery of antitumor electron microscopy, and transmission electron microscopy studies. At equal drugs due to their precisely controllable dose, mPEGylated dendrimer–DOX conjugate-based nanoparticle results in size, low polydispersity, and multiply modsignificantly high antitumor activity, and induces apoptosis on the 4T1 breast ifiable surface functionality,[11,12] with contumor model due to the evidences from tumor growth curves, an immunocomitant possibility to delivery drugs and optimize drug properties such as pharmahistochemical analysis, and a histological assessment. The in vivo toxicity cokinetics comparable to typical colloidal evaluation demonstrates that nanoparticle substantially avoids DOX-related or macromolecular delivery systems.[13,14] toxicities and presents good biosafety without obvious side effects to normal Peptide dendrimers have recently been organs of both tumor-bearing and healthy mice as measured by body weight explored as drug carriers with attractive shift, blood routine test, and a histological analysis. Thus, the mPEGylated characteristics of combining both denpeptide dendrimer–DOX conjugate-based nanoparticle may be a potential drimer and peptide, resulting in some advantages, such as water-solubility, nanoscale drug delivery vehicle for the breast cancer therapy. biodegradability, biocompatibility, and immunocompatibility.[15,16] Despite these promising features, challenges facing the in vivo antitumor 1. Introduction application of peptide dendrimer drug delivery systems have remained since the reported dendrimers with small size were Cancer remains one of the most devastating diseases; however, rapidly cleared from the circulation.[17] Although longer blood current drugs used for cancer treatments, especially chemotherapeutic drugs, kill healthy cells and cause toxicity to the circulation and higher antitumor efficacy can be achieved by patient. Nanoparticles, as drug delivery vehicles, are rapidly increased size of dendrimers by increasing the generation, progressing and have demonstrated the potential to revolusome issues such as difficult synthesis and relevant increased tionize cancer therapy by improving the treatment efficacy and toxicity would be involved.[18] reducing the side effects of chemotherapeutics.[1–3] Nanoscale To mitigate these problems, conjugating poly(ethylene glycol) (PEG) chains to the periphery of dendrimers to condrug delivery vehicles, due to the enhanced permeability and struct dendrimer-based nanoparticles is a preferred method retention (EPR) effect, have the ability to increase their accufor increasing molecular weights and size of dendrimers to mulation in the solid tumor by ineffective lymphatic drainage increase blood circulation time and tumor accumulation by and increased vascular permeability present within the tumor EPR effect while resulting in little toxicity.[19] Additionally, microenvironment.[4,5] Although a variety of nanoscale systems, PEGylation of dendrimers can assist in avoiding premature clearance by the reticuloendothelial system (RES). Thereby the Dr. C. Zhang, D. Pan, Prof. K. Luo, Dr. W. She, PEGylated dendrimer constitute an attractive platform for drug Dr. C. Guo, Y. Yang, Prof. Z. Gu National Engineering Research Center for Biomaterials delivery.[20–23] The general approach to prepare PEGylated denSichuan University drimers is coupling activated PEG chains to the end groups of Chengdu 610064, China dendrimers.[24,25] This method commonly uses amino group as E-mail: [email protected]; [email protected] a handle to introduce PEG chains, however, it is low effective and often requires long reaction time.[26] Fortunately, the highly DOI: 10.1002/adhm.201300601

Adv. Healthcare Mater. 2014, DOI: 10.1002/adhm.201300601

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efficient “click reaction,” such as the Cu(I)-catalyzed alkyne– azide click cycloaddition (CuAAC) reaction, is attractive alternative in this context, remarkably minimizing undesired side reactions and maintaining low polydispersity of dendrimers.[27] In recent study, we successfully prepared mPEGylated dendron–DOX conjugate via the CuAAC reaction, and the conjugate can self-assemble into nanoscale particles and explored as a pH-sensitive drug delivery system.[27] As drug carriers, dendrimer-based nanoparticles developed for cancer therapeutics are necessary to be capable of allowing the free drugs to be selectively released into the tumor tissues or inside the tumor cells, which can significantly enhance the antitumor efficacy and decrease side effects of drugs.[28] The enzyme-responsive drug delivery systems have been explored as the effective and selective carriers generally conjugating drugs by linkers that are only cleaved by specialized enzyme present in the tumor cells.[29–32] The enzyme of cathepsin B, a lysosomal cysteine protease overexpressed in many tumor cells and tumor endothelial cells,[33] is frequently implicated as a significant prognostic factor in primary breast cancer.[34] The glycylphenylalanylleucylglycine tetra-peptide spacer (Gly–Phe–Leu–Gly,

GFLG), as an appropriate substrate of cathepsin B,[35] has been employed in some polymer therapeutics,[36] exhibiting great stability in plasma and serum during transport and permitting intralysosomal drug liberation after endocytosis.[36,37] Based on the above-mentioned observations, our question here was if the PEGylated peptide dendrimer functionalized with peptide GFLG spacer and DOX can aggregate into nanoparticle and be suitable as nanoscale drug delivery with good biosafety as well as significant antitumor efficacy with lower dose of drug. To the best of our knowledge, however, the in vitro and in vivo antitumor applications of peptide dendrimer–drug conjugate-based nanoparticles employing GFLG linker were not studied. In this study, we described the preparation and characterization of mPEGylated peptide dendrimer–DOX conjugate-based nanoparticle as an enzyme-responsive drug delivery system for breast tumor therapy. The surface of peptide dendrimer was selectively modified with alkynyl groups and hydrosulfide groups. The enzyme-sensitive tetrapeptide (GFLG) was chosen as a linker for the conjugating the drug DOX to dendrimer via thiol-ene coupling reaction; azido mPEG chains were conjugated to dendrimer via the CuAAC reaction (Figure 1; Figures S1–S4,

Figure 1. Structures and synthesis of mPEGylated dendrimer–DOX conjugate, and the illustration of mPEGylated dendrimer–DOX conjugate-based nanoparticle.

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2. Results and Discussion 2.1. Design and Preparation of mPEGylated Dendrimer–DOX Conjugate-Based Nanoparticles Peptide dendrimers, especially the lysine-based and glutaminebased dendrimers, have been utilized as drug/gene delivery carriers and magnetic resonance imaging contrast agents due to their good biosafety in our previous studies.[18,26,38–43] For dendrimers as drug delivery vehicles, the larger size is required for the EPR effect. However, due to the small size, traditional dendrimers can be rapidly cleaned up from body.[17] Although the dendrimers with high generation showed larger size, the higher generations resulted in higher toxicity.[18] Recently, due to its excellent solubility in water, high flexibility, low protein absorption, and high biosafety, PEG was widely employed to modify drug delivery vehicles.[44,45] In our previous studies, the mPEG (2 kDa)-modified dendrimer as MRI probes showed much longer blood circulation compared to other dendrimers,[26] which was beneficial for its use as an antitumor drug delivery system. The mPEGylated dendron–DOX conjugate as a pHresponsive drug delivery system demonstrated good antitumor efficacy.[27] Therein, mPEGylated peptide dendrimer–DOX conjugate based nanoparticle was prepared as the enzyme-responsive drug delivery system for breast cancer therapy. To achieve satisfactory antitumor efficacy, most drug delivery systems were designed to be “smart,” which meant the drugs could be released from the carriers at the site of their targeted by enzymatic or chemical hydrolysis.[46] In the case of enzymatic hydrolysis, oligopeptide linker GFLG was extensively used as the stimuli part of drug delivery systems.[47,48] In most drug delivery systems with oligopeptide linker, the drug was attached to the end of spacer via amide bond.[48] In this case, the DOX–GFLG moiety was conjugated to dendrimer via a thiol-ene reaction. Mass spectrometry was employed to confirm the synthesis of dendrimer– DOX conjugate. For matrix-assisted laser desorption ionization time-of-light (MALDI–TOF MS) of alkyne–dendrimer–DOX (m/e = 6006), the most abundant peak (m/e = 6044) was assigned as [M + K]+ (Figure S5A, Supporting Information), indicating one MA-GFLG-DOX moiety was conjugated to dendrimer. After the click reaction of azido-mPEG with alkyne–dendrimer–DOX, the trace amount of copper which may cause unwanted toxicity could be removed by dialysis in ethylene diamine tetraacetic acid (EDTA) solution. The mPEGylated dendrimer–DOX conjugate was nicely purified by size-exclusion chromatography using a Superose 12 HR/10/300 GL column on an ÄKTA FPLC system and dialysis. Small molecules, excess of mPEG moiety and byproducts were easily removed. The exact mass of mPEGylated dendrimer–DOX conjugate was detected by MALDI–TOF, and the most abundant peak (m/z =


26 295) was assigned as a sodium adduct [M + K]+ (Figure S5B, Supporting Information), indicating that the average number of azido mPEG conjugated to a single dendritic molecular was 10, less than the theoretical number (12). The possible reason could be the high steric hindrance to chemical reaction, despite the click reaction with high efficiency. The yield was over 60% due to the high efficiency of click chemistry. UV–vis spectrophotometry analysis was employed to determine the content of DOX, resulting in 1.0 wt% (weight percent). The low drug content may be due to the steric hindrance of MA-GFLG-DOX while reacted with dendrimer. In the phosphate buffered saline (PBS) buffer (pH 7.4), the mPEGylated dendrimer–DOX conjugate-based nanoparticle was obtained, where the antitumor drug DOX conjugated to dendrimer via an enzyme-sensitive GFLG peptide linker. Thus, unlike other self-assemble nanocarriers such as micelles, liposomes, and polymersomes, this dendrimer-based prodrug may be stable in PBS or blood circulation because the drug is covalently linked to the dendrimer.

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Supporting Information). The in vitro and in vivo characteristics of dendrimer–DOX conjugate-based nanoparticle as the enzyme-responsive drug delivery system, such as size and morphology, zeta potential, antitumor efficacy and toxicity, were evaluated, suggesting the enzyme-responsive mPEGylated peptide dendrimer–DOX conjugate-based nanoparticle may be as a potential drug delivery vehicle for breast cancer chemotherapy.

2.2. Size and Zeta Potential of Nanoparticle Dynamic light scattering (DLS) results showed the mPEGylated dendrimer–DOX conjugate can assemble into particle with nanoscale size in water (pH 7.4), giving a size of around 122 nm (polydispersity index, PDI = 0.813) (Figure 2A). The driving force attributed to the self-assembly behavior is the minimization of the interfacial energy governed by the balance between the hydrophilic interaction of the PEG moieties and the hydrophobic interaction of the core of dendrimer.[49] Additionally, due to multiple domains of DOX, such as hydrophobic, aliphatic, and aromatic, the driving forces that governed self-assembly of our prepared dendrimer–DOX conjugate, such as π–π stacking, dipole interactions, H-bonding, and the preorganized branched architecture should contribute to the self-assemble behavior.[50] The zeta potential of the nanoparticle was −15.6 mV, suggesting that both of the strong interaction between nanoparticle and serum proteins, and being uptake by the macrophage in the circulation system could be reduced and even prevented because of the negative charge of the periphery of the carrier.[51,52] Thus, the accumulation within target tissue (tumor) in vivo of this dendrimer–DOX conjugate may be enhanced, leading to higher antitumor efficacy. As the results of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the mPEGylated dendrimer–DOX conjugate-based nanoparticle was observed (Figure 2B,C). The nanoparticles were compact, which may be due to the strong aggregation of dendrimer–DOX conjugate via noncovalent forces mediated by DOX. The sizes of particles observed by SEM and TEM were around 100 nm, which were smaller than that of those measured by DLS. That is due to the diameter of nanoparticles determined by DLS measurement is the hydrodynamic diameter of dendrimer–DOX conjugate, while that SEM and TEM image depicted the actual size of samples at the dried state. It is currently accepted that the nanoscale particles can efficiently accumulate within tumor tissues via the EPR effect.[4] In this study, our prepared nanoparticles have the diameter of 100–122 nm measured by DLS, SEM, and TEM, indicating that the dendrimer–DOX conjugate-based nanoparticle may be not only large enough to have accessibility to



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Figure 3. In vitro efficacy. 4T1 murine breast tumor cells upon incubation with free doxorubicin (DOX) and mPEGylated dendrimer–DOX conjugate-based nanoparticle (Dendrimer–DOX) at DOX-equivalent concentrations. Values represent mean ± SD (n = 3).

Figure 2. The size measured by A) DLS, B) SEM, and C) TEM of mPEGylated dendrimer–DOX conjugate-based nanoparticle. The hydrodynamic size of the particle was around 122 nm, and SEM and TEM showed around 100 nm.

tumors via EPR effect and avoid their rapid leakage into blood capillaries, but also small enough to escape capture by fixed macrophages,[1] resulting in possibility to high antitumor efficacy.

shown in Figure 3, the IC50 (half maximal inhibitory concentration) of free DOX against 4T1 cells was 0.35 µg mL−1, while that of dendrimer–DOX conjugate-based nanoparticle (IC50, 0.288 mg mL−1, being equivalent to 2.88 µg DOX mL−1) was found to be approximately eightfold of that of free drug DOX. The possible reason was the free amphipathic DOX can easily cross the cell membrane via free diffusion and be untaken by tumor cells. In contrast, the DOX linked to dendrimer via GFLG spacer can only be released after endocytosis and entering lysosome.[37] Another reason may be the slow release of DOX from the dendrimer under the intracellular environment due to the high steric hindrance of DOX (See Figure S6A and Figure S6B, Supporting Information).[53] Meanwhile, no obvious cytotoxicity was observed across the high tested concentration range (0.16–2.59 mg mL−1) (Figure S7A, Supporting Information) for the control mPEGylated dendrimers without drug, indicating the cytotoxicity of the dendrimer–DOX conjugate-based nanoparticle was only induced by the released DOX itself, not the drug-free dendrimers. As shown in Figure S6 (Supporting Information), although the drug release from nanoparticle is slow at presence of papain, released DOX was observed at absence of papain, indicating the prepared drug delivery vehicle was enzyme sensitive. Simultaneously, cytotoxicity of the drugfree dendrimers against L02 normal cells was evaluated either. As shown in Figure S7B (Supporting Information), no significant cytotoxicity was observed at the concentrations below 2.59 mg mL−1, further underlining the drug-free material was biocompatible and the dendrimer with mPEGylation can be employed as a safe drug delivery carrier. The results of obvious in vitro cytotoxicity of dendrimer–DOX conjugate-based nanoparticle demonstrated that DOX can be released from the nanoparticle to produce cytotoxicity against tumor cells, and in vivo antitumor efficacy may be achieved once the nanoparticle reach tumor tissues and enter tumor cells.

2.3. In Vitro Cytotoxicity To evaluate the cytotoxic effect of free drug DOX and mPEGylated dendrimer–DOX conjugate-based nanoparticle against the 4T1 cell line, cells were exposed to the drug formulations for 48 h, and the cell viability was tested by CCK-8 assay. As

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2.4. In Vivo Efficacy The antitumor activity and toxicity of the mPEGylated dendrimer–DOX conjugate-based nanoparticle were evaluated




FULL PAPER Figure 4. In vivo tumor growth inhibition (TGI) of mPEGylatd dendrimer–DOX conjugate-based nanoparticle. A comparison of the tumor inhibition effect of nanoparticle (being equivalent to 1 mg DOX kg−1 mouse and 4 mg DOX kg−1 mouse, respectively) versus free drug DOX (4 mg DOX kg−1 mouse) and saline in the breast tumor model (n = 5). A) The dendrimer–DOX conjugate-based nanoparticle demonstrated significant tumor inhibition at equal dose (*p < 0.01, compared to saline; $p < 0.05, compared to free drug DOX). B) At the end of this trial, tumor tissues were collected from each sacrificed animal, and the tumor growth inhibition (TGI, %) was calculated. C) During the treatment, the mice administrated dendrimer–DOX conjugate showed no significant body lost compared to saline, while DOX treatment did not (*p < 0.001, compared to free drug DOX).

on 4T1 murine breast cancer mice model. Mice bearing 4T1 tumors were treated with nanoparticle and free DOX every 4 d for 13 d, while the saline was used as control. The dose of free DOX was 4 mg kg−1, which was commonly applied in mice models. Because it was generally agreed that nanoscale drug carriers can effectively improve the antitumor efficacy,[54] dendrimer–DOX conjugate-based nanoparticle (100 mg kg−1 mouse, being equivalent to 1 mg DOX kg−1 mouse) was used in order to maximize the efficiency of DOX while minimizing DOX-induced toxicities. The higher dose of nanoparticle (400 mg kg−1 mouse, being equivalent to 4 mg DOX kg−1 mouse) was also tested. As shown in Figure 4A,B, low-dose DOXequivalent dendrimer–DOX conjugate-based nanoparticle only have moderate effect on tumor growth inhibition (TGI), which resulted in relative tumor volume of 406.3 ± 62.3% on the 17th day, and presented TGI of 44.0%. However, 4 mg kg−1 free DOX treatment had no better therapeutic effect on TGI, resulting in relative tumor volume of 388.9 ± 55.3% and presenting TGI of 45.4%. These results demonstrated the low dose of nanoparticle was as equally effective as the fourfold higher dose of 4 mg kg−1 free DOX. On the other hand, the treatment of the nanoparticle equivalent to 4 mg kg−1 of free DOX had a considerable therapeutic effect when compared with injections of either the DOXtreated group or saline control (p < 0.01 versus saline, p < 0.05 versus free drug DOX, and nanoparticle equivalent to 1 mg kg−1 of free DOX), which resulted in relative tumor volume of 289.2 ± 40.8% and represented a sharp increased TGI of 61.9%, suggesting the 400 mg kg−1 does of nanoparticle was an effective dose to inhibit tumor growth in 4T1 model and showed significantly higher antitumor efficacy than the free DOX at an equal dose. That is attributed to its ability to increasing the accumulation of nanoparticles in tumor and the enzyme-sensitive drug release features. As measured in ex vivo imaging studies, the NIRF signal was observed strong in tumor for mice treated with nanoparticle at 24 h (Figure S8A, Supporting Information). In addition, the fluorescence intensity of tumor and each organ showed that the nanoparticle-treated group had highest mean fluorescence intensity in tumor, which was significantly higher than both control group and DOX-treated group (Figure S8B, Supporting Information). These results suggested that the nanoparticle might have higher accumulation in tumor via the EPR


effect to direct drugs to tumor. Achieved high antitumor activity of the effective dose of nanoparticle also may result from its neutral charged surface and intralysosomal DOX liberation. All the results of in vivo antitumor trials demonstrated that the mPEGylation, nanoscale size, and enzyme-responsive characteristics of the nanoparticle enhanced antitumor efficacy. Simultaneous monitoring of the body weights of the treated mice throughout the experiment was used as an indication to investigate the adverse effects of the drug. As shown in Figure 4C, the changes of body weight showed very uniform body weight fluctuations and non-indicative toxicity in both low and common dose of dendrimer–DOX conjugate-based nanoparticle compared with control groups, suggesting a low degree of systemic toxicity and better drug tolerability. However, acute toxicity of mice treated with free DOX was obvious, as ≈15% loss of their initial weight was observed on day 17. These results demonstrated that no noticeable toxicity was caused by two doses of dendrimer–DOX conjugate-based nanoparticle throughout the treatment period. At the termination of the trials, histological studies were performed to detect cancerous metastases and analyze the produced toxicity. The process of tumor growth often accompanies with invasion and metastasis.[55] As shown in Figure 5, multifocal metastasis of tumor was observed in liver tissue due to the diffuse metastatic lesion consisted of neoplastic cells for mice administrated saline. In contrast, the areas of metastatic lesion were infrequent in DOX-treated mice as well as two doses of nanoparticle treated ones, further suggesting dendrimer–DOX conjugate-based nanoparticle exhibited considerable TGI and antitumor efficacy. In addition, the histological analysis showed that possible heart toxicity of the mice treated with DOX was observed due to the hyperplasia and thickening of the endocardium with acute inflammatory cells infiltration. In contrast, all the organs were normal, and no tissue or cell lesion including degeneration and necrosis was observed for the mice treated with nanoparticles. Based on weight change and the histopathology examination, the effective dose of nanoparticle was not toxic, demonstrating the significant improvements in the therapeutic index of nanoparticle over a free drug were achieved, leading to higher antitumor efficacy and lower level of side effects at an equal dose.



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Figure 5. Histological analysis for different organs of tumor-bearing mice administrated control (Saline), free drug DOX (DOX), and two dose of mPEGylated dendrimer–DOX conjugate-based nanoparticle (dendrimer–DOX) (all tissues: ×100). Our analysis showed that the free drug DOX resulted in heart toxicity due to the observed hyperplasia and thickening of the endocardium with acute inflammatory cells infiltration (A2). In contrast, organs of mice administrated saline and nanoparticle did not exhibit signs of toxicity. In saline treated group, multifocal metastasis of tumor was observed in liver tissue (B1: diffuse metastatic lesion consisted of neoplastic cells).

2.5. Immunohistochemical Studies on Treated Tumors The findings of in vivo antitumor trials suggested 400 mg kg−1 dose of nanoparticle (being equivalent to 4 mg DOX kg−1 mouse) had remarkably higher therapeutic effect. In order to further investigate the antitumor efficacy and the tumor suppression mechanism of the nanoparticle, the tumors of the treated mice were removed at the termination of the trial. The immunohistochemical studies were performed to assess cell proliferation and apoptosis in the tumors. Considering the effective dose of nanoparticle had been determined, only the tumors of mice treated with saline, free DOX, and 400 mg kg−1 dose of nanoparticle were tested. To assess the therapeutic effect of drugs on tumor cell proliferation, the immunohistochemical staining of Ki-67 was tested, which was widely used as a cell proliferation marker to stain proliferation active cells in the G1, G2, and S phases of the cell cycle.[56] As shown in Figure 6, compared to the control group, the ki-67 level of dendrimer–DOX conjugate-based nanoparticle-treated group was significantly lower (p < 0.01), indicating less Ki-67 positive cells and a low level of cell proliferation in tumor tissues. Meanwhile, statistically significance was also obtained compared to the DOX-treated group (p < 0.05). The

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examined much less active cell proliferation of nanoparticletreated group showed obviously higher antitumor activity compared to free drug DOX, consistent well with enhanced therapeutic effect on TGI. Additionally, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, one of the main methods commonly used for detecting apoptotic programmed cell death, was performed to determine apoptosis level in the tumors from different treated mice.[57] Similar to control, free drug DOX-treated tumor showed only more than ≈12% of apoptotic cells (Figure 6). In contrast, higher apoptosis level was observed in dendrimer–DOX conjugate-based nanoparticle treated ones (≈58% of apoptotic cells, p < 0.01 versus untreated and DOX-treated tumors). The immunohistochemical studies, consistent with the histological examination, demonstrated that the effective dose of nanoparticle resulted much higher treatment effect than that of equal dose of free DOX. Importantly, the nanoparticle induced a favorable prognosis due to effective inhibition of tumor cell proliferation since cancer is a disease of deregulated cell proliferation.[58] Simultaneously, differing from the anti-mitotic of most chemotherapeutic drugs acting by crudely interfering with the basic machinery of DNA synthesis and cell division, the treatment efficacy of dendrimer–DOX conjugate-based nanoparticle,




FULL PAPER Figure 6. The Ki-67 and TUNEL immunohistochemical (IHC) staining of tumor tissues. The brown areas indicate Ki-67-positive or TUNEL-positive staining. Representative photoimages of 4T1 tumors harvested at the end of study, from mice receiving various treatment with physiological saline as control (Saline), free drug DOX (DOX), and an effective dose of mPEGylated dendrimer–DOX conjugate-based nanoparticle (Dendrimer–DOX). The positive ratio of staining cells was calculated. The ki-67 density in each image was by ki-67-positive area/total area (*p < 0.01, compared to saline; $p < 0.05, compared to free drug DOX). Apoptotic index was calculated as a ratio of the apoptotic cell number to the total tumor cell number in each microscope field (*p < 0.01, compared to saline and free drug DOX). Datas are presented as mean ± SD (n = 3).

assessed by TUNEL assay, may be more effective in inducing apoptosis of tumor cells. As a novel drug delivery system, mPEGylated peptide dendrimer–DOX conjugate-based nanoparticle may be unique with much longer blood circulation time and higher accumulation in tumor tissue via the EPR effect. The nanoparticle alters the pharmacokinetics of the drug, resulting in a slower plasma clearance than free DOX, thus allowing an extravasation at the site of enhanced permeability. It should be noted that the drug DOX was covalently linked to peptide dendrimer via the GFLG peptide spacer, which is most susceptible to tumor-overexpressed lysosomal cysteine proteinase cathepsin B and stable in plasma and serum during transport,[36,37] resulting in slow elution of DOX and increased concentration of drug within the tumor,[53] which may significantly influence the environment in tumor tissues and then regulating tumor growth by inducing apoptosis of tumor cells. It has been studied that the inhibition of apoptosis lies at the heart of all tumor development,[59] inducing apoptosis may be a main reason of dendrimer–DOX conjugate-based nanoparticle to achieve much higher treatment with an equal dose of DOX.

2.6. Clinical Signs, Body Weight Changes, Hematological Analysis, and Histology on Normal Mice The possibility of nanoparticles as drug delivery carriers for clinical use depends in large part on the biosafety, which remain central concerns. To systematically assess the potential toxicity towards human, our studies based on normal mice presented the in vivo tests of the mPEGylated peptide dendrimer–DOX conjugate-based nanoparticle. In our experiments, healthy and tumor-free mice were intravenously administrated with saline (control), DOX, and nanoparticle (at equal dose), respectively, and the fluctuation in body weight and clinical signs were recorded. During the study period of 17 d, the mice administered saline and nanoparticle showed no obvious signs of


dehydration, locomotor impairment, weakness, inability to eat or drink and other symptoms associated with animal toxicity. Abnormal physical signs, behaviors, and gross toxicity were not detected, either. However, the DOX-induced toxicity of nutritional and gross metabolic was obvious. According to Figure S9 (Supporting Information), after intravenously administrated with the agents (saline, DOX, and dendrimer–DOX), body weight of mice treated with DOX did not recover to normal weight, showing about 12% weight loss compared with the control, while no abnormal body weight change was observed for nanoparticle-treated group. Those results indicated that dendrimer–DOX conjugate-based nanoparticle might produce no significant in vivo toxicity compared to drug DOX. As drug delivery vehicles, most nanoparticles designed for the further clinical use will be intravenous administration. Blood and blood components thereby may be the first physiological system interacted with nanoparticle before reaching the site of action. Thus it is important to measure whether or not the nanoparticle would induce toxicity in the blood, and the blood routine test was used for blood cell analysis. As shown in Figure S10 (Supporting Information), the important hematology markers, red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), platelet (PT), mean platelet volume (MPV), and white blood cell (WBC) count were tested. All of the above parameters for nanoparticle-treated group were in normal range without physiologically significant difference in comparison with the control group, suggesting no syndromes including hemolytic anemia, acute infection, and bone marrow dysfunction were caused by nanoparticle. Furthermore, H&E-stained sections of the main organs were examined to further investigate any potential toxicity of dendrimer–DOX conjugate-based nanoparticle, such as tissue damage or inflammation. As presented in Figure S11 (Supporting Information), a histopathology examination did not reveal signs of toxicity for dendrimer–DOX conjugate-based nanoparticle-treated group. However, for free DOX-treated



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group, the heart toxicity may be induced by DOX due to the observed hyperplasia and thickening of the endocardium with acute inflammatory cells infiltration (Figure S11, Supporting Information). The non-observed toxicity of mPEGylated peptide dendrimer–DOX conjugate-based nanoparticle could result from its lower molecular weight, optimized surface properties of PEGylation, and the biodegradability of lysine-based dendrimer.[27] Coating of the nanoparticle with PEG can play an important role in evading the immune system and associated phagocytes to avoid inducing unacceptable toxicity through the activation of immune responses. The biodegradability of peptide dendrimer can also promote its clearance from the organism and thereby enhance the in vivo biosafety.[60] The drug-loaded nanoparticle may have the ability to higher accumulate in tumor tissue but lower accumulate in normal tissue via the EPR effect, which further reduced the side effects to normal organs.[5] In addition, the tetra-peptide linker GFLG in dendrimer–DOX conjugate-based nanoparticle was designed to only permit DOX released from the delivery vehicle in tumor cells by cathepsin B, which is overexpressed in tumor cells. Thus, the mPEGylated peptide dendrimer-based nanoscale drug delivery system achieved in vivo tumor treatment efficacy with minimal toxicity.

3. Conclusions We have described an example of mPEGylated peptide dendrimer drug conjugate-based nanoparticle as an attractive platform for drug delivery with excellent characteristics and functionalities. The anticancer drug DOX was conjugated to the mPEGylated peptide dendrimer via a tetra-peptide linker GFLG, which was sensitive to cathepsin B overexpressed in the tumor cells. Combining the features of peptide dendrimer and nanoscale materials, dendrimer–DOX conjugate-based nanoparticle was found to have obviously improved in vivo antitumor efficacy over commercial DOX formulation at equal dose. Even a low dose of nanoparticle has equivalent antitumor efficacy as the common dose-free DOX. Such unique dendrimer–DOX conjugate-based nanoparticle also provided an opportunity for breast cancer therapy due to the less toxicity as measured by acute changes in body weight, blood cell counts, and histological analysis. Therefore, the antitumor efficacy of dendrimer–DOX conjugate-based nanoparticle also can be further improved via increase of the drug loading and optimization of the dose. Further studies will focus on higher drug loading and higher concentration of dendrimer–DOX conjugate-based nanoparticle. Overall, the structural design of mPEGylated peptide dendrimer–DOX conjugate-based nanoparticle in this study may provide useful strategies for design and preparation of peptide dendrimer as safe and effective drug delivery systems.

4. Experimental Section Materials and Measurements: N,N-Diisopropylethylamine (DIPEA), 5-hexynoic acid, 1-hydroxybenzotriazole (HOBt), N,N,N′,N′-tetramethyl-(1Hbenzotriazol-1-yl)uronium hexafluorophosphate (HBTU), trifluoroacetic

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acid (TFA), methoxy poly(ethylene glycol) (mPEG, 2 kDa), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were purchased from Sigma– Aldrich and used without further purification. Boc-L-Lys(Cbz)-OH, Boc-L-Lys(Boc)-OH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), and 3-tritylsulfanylpropionic acid (TFA) were purchased from GL Biochem (Shanghai) Ltd. Boc- and Cbz-protected dendrimer,[26] azido methoxy poly(ethylene glycol) (azido-mPEG),[61] and N-methac ryloylglycylphenylalanylleucylglycyl-doxorubicin (MA-GFLG-DOX)[48,62] were prepared as previous described. Characterization and structural confirmation of dendritic intermediates and products were performed by 1H NMR, electrospray ionization–mass spectrometry (ESI–MS, TSQ Quantum Ultra LC–MS/MS), and matrix-assisted laser desorption ionization–time-of-light (MALDI–TOF, Autoflex MALDI–TOF/TOF) MS. 10 mg mL−1 2,5-dihydroxybenzoic acid (DHB) (water/acetonitrile = 2/1, 0.1% TFA) and mixture of 125 µL of a diammoniumhydrogen citrate distilled aqueous solution (18 mg mL−1) or 375 µL ethanol solution of 2,5-dihydroxyacetophenon (DHAP) (20.2 mg mL−1) were used matrix solution for sample preparation for MALDI analysis. 1H NMR data were obtained using a 400 MHz Bruker Advanced Spectrometer at room temperature, and chemical shifts are reported in ppm on the δ scale. DLS and zeta potential measurements were performed in MilliQ water using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). All samples of 100 mg mL−1 were measured at 20 °C. The samples were prepared by directly dropping the solution of nanoparticle onto slice of silicon and dried at room temperature overnight for SEM (S-4800, HITACHI, Japan) studies. The Synthesis of Alkyne–Dendrimer–DOX: The details of the synthesis of alkyne–dendrimer and alkyne–dendrimer–STrt were described in the Supporting Information. The alkyne–dendrimer-STrt (1.20 g, 0.15 mmol) was dissolved into anhydrous DCM/TFA (1:1, 8 mL) and the solution was stirred for 0.5 h at 0 °C under nitrogen. When the solution turned to yellow-green, triethylsilane (145.6 mg, 1.25 mmol) was added into the flask. The solution was stirred for another 24 h at room temperature under nitrogen (Figure S3, Supporting Information). After the solvent was removed by rotary evaporation, diethyl ether was added and white precipitates appeared. The precipitate was collected by centrifugation and washed three times with anhydrous diethyl ether. The sample was dried under high vacuum for 0.5 h and dissolved into 20 mL anhydrous DMF followed by MA-GFLG-DOX (1.89 g, 1.89 mmol), DBU (2.74 g, 18 mmol). The solution was stirred for 72 h at room temperature under nitrogen in darkness. After woke up, the mixture was added into 300 mL EtOAc by dropwise, and red precipitate appeared. The precipitate was collected by centrifugation and purified by sizeexclusion chromatography using a Superose 12 HR/10/300 GL column on an ÄKTA FPLC system (GE Healthcare) column with sodium acetate buffer containing 30% acetonitrile (pH 6.5) as mobile phase. The residue was dried under high vacuum. MALDI-TOF MS: m/z 6044 [(M+K)+, C290H449N51O61S12K]. The Synthesis of mPEGylated Dendrimer-DOX Conjugate-Based Nanoparticle: Under nitrogen, alkyne–dendrimer–DOX (150 mg, 25 µmol), CuSO4·5H2O (90 mg, 0.36 mmol), N3-mPEG (1.1 g, 0.54 mmol), and sodium ascorbate (142 mg, 0.72 mmol) were added into the 20 mL mixture of DMF and H2O (3:1, v/v). The mixture was stirred for 3 d in darkness (Figure S4, Supporting Information). After woke up, the solution was dialyzed under darkness. The solvent was removed by freeze-drying and the residue was further fractionated/purified by sizeexclusion chromatography using a Superose 12 HR/10/300 GL column on an ÄKTA FPLC system (GE Healthcare) column with sodium acetate buffer containing 30% acetonitrile (pH 6.5) as mobile phase. After dialysis against water in darkness and freeze-drying, the final product was obtained in 75% yield (45.8 mg). MALDI-TOF MS: m/z 26295 ([M + K]+) Size, Shape, and Zeta Potential: The hydrodynamic size and zeta potential of the nanoparticle were characterized using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). The mPEGylated dendrimer–DOX conjugate was diluted to 10 mL with distilled water to a final concentration of 100 µg mL−1. The TEM samples (concentration of 100 µg mL−1) were prepared by dipping a copper grid with formvar film into the freshly prepared nanoparticles solution. A few minutes after the deposition, the aqueous solution was blotted away with a strip of filter paper and then the samples were dried at room temperature. The




Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements C.Z. and D.P. contributed equally to this work. The work was supported by National Natural Science Foundation of China (51133004, 81361140343,


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size of mPEGylated dendrimer–DOX conjugate-based nanoparticle was observed by field emission scanning electron microscope (FE-SEM). The samples (concentration of 100 µg mL−1) were prepared by directly dropping the solution of sample onto slice of silicon and dried at room temperature overnight for SEM studies. Tumor Cell Lines, Cell Culture, and Animals: 4T1 cell line (a cell line was derived from the BALB/c spontaneous mammary carcinoma) and human liver cell lines (L02) were purchased from Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China). The cell line was maintained as monolayer cultures in RPMI 1640 medium (Life Technologies), supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS, Hyclone), 1% penicillin and streptomycin mixture, and incubated in 5% CO2/95% air at 37 °C. Female BALB/c mice (20 ± 2 g, 6–8 weeks old) were selected for tumor model. The animals were purchased from West China Animal Culture Center of Sichuan University, which were randomly divided and housed in a controlled temperature room with regular alternating cycles of light and darkness. All animals were performed in line with national regulations and approved by the animal experiments ethical committee. In Vitro Cytotoxicity Assays: Cytotoxicity of the drug-free dendrimer, dendrimer–DOX conjugate-based nanoparticles, and free DOX were evaluated by the percentage of cell viability via Cell Counting Kit-8 (CCK8). 5.0 × 103 cells/well 4T1 cells and L02 cells were seeded in 96-well plates. After 24 h culture, the drug-free dendrimer, dendrimer–DOX conjugate-based nanoparticles and free DOX solutions containing RPMI 1640 culture medium were added to each well, respectively. After incubation for further 48 h, 10 µL CCK-8 was added to each well and the plates were incubated at 37 °C for another 2 h. Then, the absorbance of each sample was measured using a microplate reader Varioscan Flash (ThermoFisher SCIENTIFIC). The cell viability (%) was obtained according to the manufacturer’s instruction. In Vivo Efficacy: In vivo antitumor efficacy was investigated in subcutaneous 4T1 model. To produce tumors, female BALB/c mice were inoculated with 4T1 suspension via the following procedures. Mice were anesthetized and then 5 × 105 4T1 cells suspended in 50 µL PBS were subcutaneously injected into the previously treated region of each mouse. Solid tumors were allowed to form over a period of 1–2 weeks to reached a volume ranging from 50 to 100 mm3, then tumorbearing mice were randomized to receive different treatment with saline (control), DOX (4 mg DOX kg−1 mouse) and two doses of dendrimer– DOX conjugate-based nanoparticle (being equivalent to 1 mg DOX kg−1 mouse and 4 mg DOX kg−1 mouse, respectively) in a final volume of 200 µL via the tail vein every 4 d for four times. The tumor sizes and the mouse body weights were measured every 2 d. Tumor volumes and body weights were normalized to 100% at day 0. The tumor volumes were calculated by the following formula: tumor volume V (mm3) = 1/2 × length (mm) × width (mm2). At day 17, all of the animals were euthanized and sacrificed, the tumors were excised for immunohistochemical study to evaluate the angiogenesis, apoptosis, and cell proliferation in tumor. Tissues and organs, such as liver, heart, spleen, lung, and kidney were separated and fixed for histological examination. Statistical Analysis: A comparison between groups was analyzed by the Student’s t -test was used to address statistical significance. All data are presented as mean ± SD, with p values of

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