Enzyme-Sensitive and Amphiphilic PEGylated Dendrimer-Paclitaxel

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Enzyme-Sensitive and Amphiphilic PEGylated Dendrimer-Paclitaxel Prodrug Based Nanoparticles for Enhanced Stability and Anticancer Efficacy Ning Li, Hao Cai, Lei Jiang, Jiani Hu, Ashika Bains, Jesse Hu, Qiyong Gong, Kui Luo, and Zhongwei Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15505 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on January 23, 2017

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ACS Applied Materials & Interfaces

Enzyme-Sensitive and Amphiphilic PEGylated Dendrimer-Paclitaxel Nanoparticles

for

Prodrug Enhanced

Based

Stability

and

Anticancer Efficacy Ning Li,†,‡ Hao Cai,† Lei Jiang,‡, Jiani Hu,§ Ashika Bains,§ Jesse Hu,§ Qiyong Gong,† Kui Luo*,† ∥

and Zhongwei Gu*,†,#, †

∥

Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital,

Sichuan University, Chengdu 610041, China ‡

State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for

Metabolic Diseases,

Center of Advanced

Pharmaceuticals and

Biomaterials, China

Pharmaceutical University, Nanjing 210009, China #

College of Materials Science and Engineering, Nanjing Tech University, Nanjing 211816,

China ∥

National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064,

China §

Department of Radiology, Wayne State University, Detroit, MI 48201, USA

Corresponding author : Fax: +86 28 85410653; Tel: +86 28 85410336/+86 28 85410653 E-mail: [email protected]; [email protected]

ACS Paragon Plus Environment

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ABSTRACT: In this study, we prepared a smart polymeric vehicle for the hydrophobic drug paclitaxel (PTX) which allowed a maximum steady state circulation and a fast intracellular release in tumors. PTX was linked to the Janus PEGylated peptide dendrimer via an enzymesensitive linker glycylphenylalanylleucylglycine (GFLG) tetra-peptide by efficient click reaction, resulting in Janus dendritic prodrug with 20.9% PTX content. The prodrug self-assembled into nanoscale particles with appropriate nano-sizes, compact morphology, and negative surface charge. In addition to high stability during circulation, as demonstrated by protein adsorption assays and drug release studies in the cancer’s intracellular environment, the nanoparticles were able to quickly release the drug intact in its original molecular structure, as verified via high performance liquid chromatography and mass spectrometry analyses. Compared to free PTX, the enzyme-responsive feature of nanoparticles promoted higher cytotoxicity against 4T1 cancer cells and much lower cytotoxicity against normal cells. The nanoparticles accumulated in the tumor, and were retained for an extended period of time, as confirmed by fluorescence imaging. Therefore, these nanoparticles exhibited significantly enhanced antitumor efficiency in the 4T1 breast cancer model as indicated by the observed inhibition of angiogenesis and proliferation as well as induction of apoptosis. Moreover, the nanoparticles reduced the occurrence of side effects, particularly dose-limited toxicities, as monitored by body weight and hematological features. Hence, our Janus PEGylated dendrimer-PTX prodrug-based nanoparticles may potentially serve as nanoscale vehicles for breast cancer therapy. KEYWORDS: Janus dendrimer; prodrug; enzyme-sensitive; drug delivery; stability


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1. INTRODUCTION Chemotherapy faces several challenges such as high biodistribution of chemotherapeutic agents into normal tissues leading to adverse side effects and low tumor accumulation resulting in unsatisfactory anticancer efficacy. Numerous strategies have been employed to improve the efficiency of anti-cancer drugs. These strategies are collectively described using the abbreviation SAIR (Stability, Accumulation, Internalization and Release),1-2 which include improving the stability of drugs during circulation (Stability),3 enhancing drug accumulation in tumors through active or passive targeting (Accumulation),4 promoting drug internalization into tumor cells (Internalization), and accelerating intracellular drug release (Release).5 Some of these strategies have been determined to enhance some specific drug feature, but fail to provide improvement of the overall anticancer efficacy. Paclitaxel (PTX) is one of the most effective cytotoxic anti-tumor drugs. However, it is rapidly eliminated from the circulatory system and induces side effects, such as marrow toxicity, thus limiting its application.6 Moreover, PTX is degraded in the circulation system through cleavage of its ester bonds.7 Based on these factors, multifunctional nanoscale drug delivery carriers provide the benefit of mitigating limitations of PTX therapy by utilizing the SAIR strategy, thereby allowing for enhanced therapeutic efficiency and lower side effects. Among the various reported carriers, peptide dendritic polymers, as macromolecules prepared from amino acids, have well-defined nanoscale sizes and flexible surface chemistry.8-9 These features make such macromolecules potential candidates for drug delivery.10-12 Moreover, Janustype dendritic polymers, which combine two chemical functionalities in their cores and inner layers, could assume different tasks. The Janus-type dendritic polymers that carry different functionalities in a single molecule offer greater versatility compared to the more common


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dendritic polymers.13 However, the dendrimer or dendron (low generation) based vehicles are < 10 nm in diameter, which is not large enough to meet the standard of enhanced permeability and retention effect (EPR effect). Although high generation dendritic polymers have slightly higher size, they are slowly degraded during circulation, thereby increasing the risk of cytotoxicity.14 PEGylation of dendritic polymers has been employed to address this issue, which resulted in prolonged blood circulation time.15 Previously, we modified low generation dendrimers or dendrons using low-molecular weight poly(ethylene glycol) (PEG, MW = 2,000) followed by drug conjugation.16 The PEGylated and drug-conjugated dendritic polymers have nanoscale sizes, long blood circulation time, and therefore high accumulation into tumors. Additionally, when the drug is hydrophobic, the PEGylated, amphiphilic and dendritic conjugates could selfassemble into nanoscale systems, and the drugs were in the core of the nanoparticles and predicted from interaction with protein in the blood circulation system, resulting in enhanced therapeutic indices.17 Intravenous (i.v.) injection of nanoparticles generally results in its adsorption by plasma proteins through a process known as opsonization.18-19 Current methods to slow or reduce opsonization have focused on introducing hydrophilic groups or modifying the particle to neutralize the surface charge.20-22 Polyethylene glycol (PEG), which can enhance the solubility and stability of plasma proteins, as well as reduce immunogenicity, was selected and utilized in the present study to modify the nanoscale carriers with the goal of both decreasing their nonspecific cellular uptake by the mononuclear phagocyte system (MPS) and modulating the circulation half-life of nanoparticles.19 Previous studies have demonstrated that PEGylated low generation dendrimers have longer blood circulation time and show enhanced tumor accumulation, signifying that PEGylated dendritic drug delivery vehicles confer significant anti-


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cancer efficacy.16-17 However, high PEG surface density in drug delivery vehicles has also been shown to hinder tumor cell uptake.3 The PEG density of nanoparticles was optimized to balance between minimizing opsonization during circulation and relatively enhancing cellular uptake within tumors. Thus, a relatively low surface PEG density was chosen to protect our nanoparticles during circulation in combination with rapid cellular internalization following accumulation within the tumor. In addition to the high accumulation rate into tumors and cellular uptake, rapid drug release from carriers is also an important feature of an ideal drug delivery system.23 Stimuli-responsive vehicles have been one of the major research topics relating to the tumor microenvironment, as the rapid release of the cargo is necessary upon uptake of nanoparticles by cancer cells.10, 24 Enzyme-sensitive polymeric drug delivery systems have been designed and studied, with demonstrated potential for clinical applications.25-26 The enzyme, for example, cathepsin B as a lysosomal cysteine protease which is overexpressed in various tumor cells such as breast cancer, has been a target in the design of enzyme-sensitive drug delivery vehicles.27-29 The oligopeptide glycylphenylalanylleucylglycine (Gly-Phe-Leu-Gly; GFLG) has been utilized to link the antitumor drugs to the polymeric carriers,30-33 and these polymer-drug conjugates have been shown to have good stability in the bloodstream, particularly during circulation, as well as the ability to release the drug in the intracellular environment by cathepsin B degredation.34-36 We previously reported that PEGylated dendrimers and their nanoscale vehicles carrying Doxorubicin (DOX) may be potentially used in cancer therapy because of the presence of dendritic structures, which enhance anti-cancer efficacy.10 However, the ultimate challenge is to integrate all necessary functions of SAIR into a single dendritic system. Based on the above observations, the present study investigated whether optimized dendritic structures and


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compositions, as well as multiple ester bond vehicles for loading PTX, undergo rapid tumor cellular internalization and fast release. In addition, the present study also examined whether the conjugate self-assembled into nanoscale vehicles was suitable as efficient and safe drug delivery carriers. In the present study, we designed and prepared amphiphilic Janus PEGylated dendrimerGFLG-PTX prodrug with a structure similar to block polymers, and assessed its potential as an enzyme-sensitive nanoscale vehicle for cancer therapy. Figure 1 shows the synthesis route of the dendritic conjugate and its potential to self-assemble into nanoparticles. Through high efficient click reaction, the enzyme-sensitive tetra-peptide GFLG-PTX moiety was attached to the PEGylated peptide dendrimer. The size (hydrodynamic state or dried state), morphology, and zeta potential of prodrug-based nanoparticles were measured through DLS and AFM. Protein adsorption experiments were conducted, and the enzyme-sensitive drug release features of the prodrug were evaluated. A series of in vitro studies such as cell uptake, cytotoxicity, and flow cytometry method (FCM) using different cell lines were performed. Additionally, the details of these prodrugs in tumors and normal tissues were evaluated using in-vivo and ex-vivo fluorescence imaging. Finally, the in vivo anticancer efficacy and biosafety of the prodrug-based nanoparticles were studied well. 2. EXPERIMENTAL SECTION 2.1. Materials and Measurements Azido-Cy5.5 was purchased from Lumiprobe (Supporting Information, Figure S1, Hallandale Beach, Fl, USA). N,N-Diisopropylethylamine (DIPEA), N,N,N',N'-tetramethyl-(1H-benzotriazol1-yl)uronium hexafluorophosphate (HBTU), and 1-hydroxybenzotriazole (HOBt) were purchased from GL Biochem (Shanghai, China). Sodium ascorbate, 5-hexynoic acid, methoxy


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poly(ethylene

glycol)

(mPEG,

2kDa),

dicyclohexylcarbodiimide

(DCC),

4-

dimethylaminopyridine (DMAP), papain, and cathepsin B were purchased from Sigma-Aldrich (St. Louis., MO, USA) and used without further purification. Dendrimer Generation 2 (Lys) and Generation 3 (Lys) are abbreviated as G2L and G3L, respectively. PEGylated dendrimer G2LG3L modified with alkyne (PEG-G2L-G3L-alkyne) and azido-GFLG derived paclitaxel (N3GFLG-PTX) (see Supporting Information) were synthesized as previously described.17,

25, 37

Mass spectrometry (ESI-MS and MALDI-TOF-MS) were used to characterize the structure of prepared products. The hydrodynamic size test was conducted in di-water or PBS on a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). In-vivo and ex-vivo fluoresce imaging were carried out on a Maestro In-Vivo Imaging System (Cri, USA). Size exclusion chromatography (SEC) was performed to purify the products using an ÄKTA FPLC system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). HPLC was perform using Zorbax C8 4.6 × 150 mm columns (Agilent 1260 LC, Santa Clara, California, USA). 2.2. Synthesis of Janus PEGylated Peptide Dendrimer-PTX Prodrug Compound N3-GFLG-PTX and Janus PEGylated dendrimer G2L-G3L-alkyne were synthesized (see Supporting Information). The Janus PEGylated dendrimer (540 mg, 50 µmol), N3-GFLG-PTX (680 mg, 0.5 mmol), and sodium ascorbate (158 mg, 0.8 mmol) were then added into a flask under an argon atmosphere. To this flask, a solution of H2O/DMSO (1:3, 100 mL) containing CuSO4·5H2O (100 mg, 0.4 mmol) was added. After stirring at 50oC for one day, the solvent, catalyst, and unreacted raw material were mostly removed through dialysis with EDTANa2 (1 mM) aqueous solution at cold temperature (4oC). After exhaustive dialysis, the solution was collected and lyophilized, yielding a pale yellow solid. The crude product was further


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purified by SEC on an FPLC system to remove unreacted organic compounds. After dialysis in water, the product was collected by freeze-drying (846 mg, 91.2% yield). Under an argon atmosphere, azido-Cy5.5 (5 mg, 5 µmol) was reacted with product above (500 mg, 28 µmol), and Cy5.5 was conjugated to the dendrimer via click reaction, as described above. The final product (Janus PEGylated dendrimer-GFLG-PTX Prodrug) was obtained at a mass of 420 mg, resulting in an 83.3% yield, MS: m/z 18471.7 [M+H]+. The content of Cy5.5 in this prodrug was approximate 0.5%wt (weight percent) as determined by fluorescence spectroscopy, and had 20.9%wt drug (paclitaxel) content as determined by HPLC. 2.3. Physicochemical Properties of Prodrug-Based Nanoparticles The purified prodrug was measured through DLS and AFM. The hydrodynamic size and surface charge of our prodrug-based particles (1 mg/mL) in di-water or PBS (pH 7.4) were measured through DLS in triplicate. Meanwhile, solution was diluted and dried for AFM test. After dried onto ultra-clean mica sheet, AFM sample was prepared. The size and morphology of the sample was then observed through AFM using an Asylum Research MFP-3D-Bio (Digital Instruments, Inc., Santa Barbara, CA, USA). The real-time size and morphology of the prodrug under various conditions were monitored by DLS and SEM. Ultrapure water was used to prepare buffers (PBS and McIlvaine’s buffer). The PEGylated dendrimer-GFLF-PTX was dissolved in the buffer (pH 7.4 or 5.4) to form nanoparticles. The prepared prodrug was incubated in the McIlvaine’s buffer (1 mg/mL, pH = 5.4) with papain (2.0 µM) for 6 h.29 Meanwhile, the prodrugs were incubated in different buffers without papain (pH 7.4 and 5.4) at 37oC for 24 h. The size and zeta potential were measured by using DLS at different pre-determined time points. At the specific time points (0 h, 1 h, 4 h, and 6 h), the diluted sample (200 µg/mL, 20 µL) was dropped onto a silicon slice for SEM tests.


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2.4. Drug Release As reported, papain and cathepsin B have similar activity. Thus, the drug released from the PEGylated dendrimer-GFLG-PTX prodrug-based nanoparticles was conducted in the presence of papain. Equal volumes of glutathione (10 mM) in McIlvaine’s buffer (pH 5.4) and enzyme solution were mixed and shaken at 37oC, and the prodrug-based nanoparticles were immediately added to the solution, as previous reported.25, 29, 32-37 The solution was incubated at 37oC for 48 h, with the concentration of papain at 2.0 µM and the concentration of nanoparticles at 3 mg/mL. Meanwhile, the prodrug-based nanoparticles (3 mg/mL) were incubated in PBS (pH 7.4 and 5.4) without papain. At pre-determined time points, the solution was drawn, diluted with methanol, and finally measured by RP-HPLC. The flow rate was set as 1.5 mL/min within 20 min (Buffer A: acetonitrile, Buffer B: ultrapure water; Buffer A/B=1:1). Simultaneously, the released segment was collected and analyzed via ESI-MS. 2.5. Cells and Animals Experiments The 3T3 (murine fibroblasts cell), C2C12 (murine myoblast cell) and 4T1 (murine breast cancer cell) cell lines were from the Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China). The studies were carried out in an incubator at 37oC (5% CO2). The 4T1 cell line was cultured with RPMI-1640, while the 3T3 and C2C12 cell lines were cultured with DMEM. For our tumor models, female BALB/c mice and nude mice (20 ± 2 g, 6–8 weeks old) were chosen and purchased from DaShuo Biological Technology Co., Ltd (Chengdu, China). All the experiments were approved by the animal experiments ethical committee and carried out according to regulation of the Sichuan University ethics committee and national regulations. 4T1 cells (1×106) in PBS (80 µL) were injected into mouse subcutaneously. About one week later, solid tumors with volumes of approximately 60 mm3 were observed. Then, three


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unified groups of inoculated mice were prepared (n = 7 each group) for in vivo anticancer and imaging studies. 2.6. In Vitro Cytotoxicity Cells (4T1, 3T3, and C2C12) were seeded in plates (96-well). After 24 h, the cells were incubated with PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles and free PTX in a special culture medium (RPMI-1640/DMEM) (100 µL per well), while untreated cells were used as control (n = 5). After incubating for 48 h, the PEGylated dendrimer-GFLF-PTX prodrug and free PTX were cleared. The cell viabilities were measured by a CCK-8 kit (Dojindo Molecular Technologies, Inc., Japan). Approximately 100 µL of the special culture media (without FBS and penicillin/streptomycin) containing CCK-8 (10%) was added for further incubation at 37oC. After 2 h incubation, the absorbance was measured and the cell viability (%) was calculated. 2.7. Cellular Uptake and Apoptosis Analyses The 4T1 cellular uptake studies were conducted through confocal laser scanning microscopy (Leica TCP SP5). Cyanine 5.5 (Cy5.5), as a near-infrared (IR) fluorescence-emitting dye, can be directly observed using fluorescence optics. Thus, the Janus PEGylated dendrimer-GFLG-PTX prodrug-based nanoparticles were conjugated with Cy5.5 to trace their behaviors in vitro and in vivo. Cells were seeded on a confocal dish. After one day, prodrug based nanoparticles (PTX concentration: 20 µg/mL) were added to the dish and incubated with the cells for 2 h. After removing the culture media and washing three times with PBS, the cells were stained with a lysosome tracker for 45 min. The cells were then washed with PBS and observed. The cells were seeded into 6-well plates (1.5 × 105 cells/well) and incubated for 24 h. The cells were then incubated with PTX injection and PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles (0.75 µg/mL PTX) for 48 h while PBS as control (n = 3). Following incubation, the


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cells were collected, and apoptotic cells were detected by FCM using the Annexin V-FITC apoptosis detection kit I (DOJINDO, Japan), according to the protocol provided by the manufacturer. These results were characterized using the WinMDI 2.9 software. 2.8. Protein Adsorption Assay To study the protein adsorption of the prodrug-based nanoparticles, bovine serum albumin (BSA) was chosen as the model protein. The nanoparticles (0.15 mg/mL) were co-incubated with BSA (0.25 mg/mL) in PBS (pH 7.4) at 37°C. At different time points, 200-µL aliquots of each sample were centrifuged (13,000g, 15 min) to promote protein-adsorbed aggregates precipitate. Then, according to the specification, BSA standard curve was established by using a BCA Protein Assay Kit (BD, Rockford IL, USA). Meanwhile, in the same condition, concentration of the protein that had not been adsorbed was measured through microplate reader (Thermo Scientific, USA). The ratios of adsorbed protein at different time points were then calculated. 2.9. In Vivo Efficacy Clinical PTX agent and prodrug-based nanoparticles were injected intravenously into 4T1 tumor-bearing mice with a PTX dose of 5 mg/kg body weight each day for 10 days. Saline was injected for control subjects (n = 7). The body weights of mice and tumor volumes were measured every other day. At the 19th day, the mice were sacrificed. Then, TGI was obtained as formula according the weight of the excised tumors. For all of the tumors, apoptosis, cell proliferation and angiogenesis were assayed via immunohistochemical (IHC) analysis of Ki-67, CD-31 and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling), as shown in our previous report.16-17 2.10. Imaging Studies


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The in vivo distribution of prodrug-based nanoparticles in the nude tumor-bearing mice was evaluated using in-vivo and ex-vivo imaging. After establishment of the breast cancer model, the female nude mice were i.v. injected with nanoparticles labeled with Cy5.5 (with a dose of 5 mg/kg PTX). Saline was injected as control. In vivo imaging of the whole body was performed at different time points after injection. After in vivo imaging, the mice were sacrificed. The tumor tissues and normal organs were separated for histological assessment using the IVIS imaging system. There were three mice per group at all time points. 2.11. In Vivo Toxicity Studies Three groups of healthy and normal mice were marked clearly (n = 7). Then, the mice were injected intravenously via the tail vein with PTX dose of 5 mg/kg body weight each day for 10 days. The body weight of each mouse was measured every other day, while the clinical symptoms of the animals were monitored daily. After 19 days, the mice were sacrificed and blood was collected for analysis. 2.12. Statistical analysis All data are expressed as mean ± SD. Statistical significance (p values of < 0.05) was calculated via student’s t-test. 3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of PEGylated Dendrimer-GFLG-PTX Prodrug To integrate the necessary functions of SAIR into a single dendritic scaffold, amphiphilic dendritic polymer-PTX prodrug was designed and prepared. To efficiently prepare the functionalized dendritic conjugate, an efficient click reaction was applied to the synthesis of the PEGylated dendrimer-GFLF-PTX prodrug. First, the PEGylated dendrimer (Janus dendrimer PEG-G2L-G3L-alkyne) was prepared with MS: m/z = 10,672.7 (see Supporting Information


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Figure S2). Second, the moiety, enzyme-responsive linker GFLG modified with azido group and drug PTX (azido-GFLG-PTX), was conjugated to PEGylated dendrimer (PEG-G2L-G3Lalkyne) via click reaction. Finally, through click reaction, Cy5.5 was linked to the dendrimerPTX conjugate. Then, after further purification via SEC, PEGylated dendrimer-GFLF-PTX prodrugs were generated and collected. The mass of the prodrug was characterized, giving a most abundant peak (m/z = 18,471.7 [M+H]+) (Figure 2) which suggested that the PEGylated dendrimer was covalently linked with approximately five azido-GFLG-PTX ligands on average. HPLC was also utilized to determine drug content (20.9 wt%), coinciding with the result of MS. Due to the conjugation of the drug PTX to the PEGylated dendrimer, the dendritic conjugate may be stable in serum and plasma, or during transportation. 3.2. Physicochemical Properties of Prodrug-based Nanoparticles The morphology and size of particles affect the in vivo distribution of materials. Previous studies have shown that a suitable size and morphology could facilitate the accumulation of a drug in a tumor.10,

20, 25

Thus, via DLS and AFM, the size and morphology of PEGylated

dendrimer-GFLF-PTX prodrug-based nanoparticles was observed. Figure 3A shows the sample aggregating into nanosized particles in water, with an average hydrodynamic size of around 69 nm, and the size distribution (PDI = 0.230) was narrow. AFM was also conducted for morphological analysis of the samples. As shown in Figure 3B, nanoparticles were observed to have uniform spherical morphology and diameters of approximately 74 nm. The diameter was close to that measured by DLS. The size measured by AFM was in agreement with those calculated by DLS. The nanoscale size tested by DLS and AFM indicated that the PEGylated and amphiphilic dendrimer-PTX prodrug could assemble into uniformly sized nanoparticles. Several strategies have been designed to fabricate nanoscale vehicles, including self-assembly. It has


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been noted that dendrimers, particularly hydrophilic ones, are incapable of engaging in chemical or physical interactions; thus the self-assembly behavior is likely governed by PEGylated dendrimer-GFLF-PTX itself. The driving force for self-assembly may be attributable to the reduction in interfacial energy, balancing the hydrophobic dendrimer-GFLF-PTX block and the hydrophilic PEG ligands. In addition, other factors, including π-π stacking and H-bonding may have influenced the self-assembly of the conjugates,

10, 17

since the GFLF-PTX moiety has

aromatic and hydroxyl groups. Of note, smaller particles more rapidly penetrate tumors compared to larger ones, however extremely minute particles could easily undergo hepatobiliary and renal clearance. It is therefore challenging to find a balance between maximizing tumor penetration (by reducing nanomedicine size) while minimizing both toxicity to normal tissue and clearance by the MPS (by increasing nanomedicine size). As reported, particles < 100 nm and > 20 nm could be a good choice.38-39 Thus, our nanoparticles of around 60–80 nm may be potentially used as vehicles with prolonged circulation time and efficient tumor penetration. An ideal drug delivery system should accumulate into a tumor after its circulation in the bloodstream. The zeta potential of nanoparticle plays a key role in its circulation in blood. The zeta potential of the PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles was negative (Figure 3C, -16.9 mV). Similar to the findings of previous studies, intravenously administered positively charge nanoparticles are rapidly cleared from circulation by the macrophages or RES. In contrast, negatively charged and neutral nanoparticles theoretically reduce protein interaction and binding, thus resulting in a longer blood half-life.19 Thus, high accumulation into tumors could be mediated by the prodrug-based nanoparticles through the EPR effect as the optimized size and negative surface charge. 3.3. Drug Release and Changes of Nanoscale Systems


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The slow release of a drug limits the anti-cancer efficacy of nanoparticles for cancer therapy. Upon internalization of a nanoparticle-based drug delivery system by a tumor cell, a rapid release of drug content is optimal. We developed a smart conjugate-based nanoparticle that may be triggered by intercellular cathepsin B overexpressed in tumor cells. Therefore, it is essential to evaluate the capacity of these vehicles to respond to stimuli. For possessing a similar activity as cathepsin B, the enzyme papain was selected for the evaluation of enzyme-responsive drug release behavior. For the copolymer-GFLG-drug conjugate, the drug may be released from the carriers due to the potential cleavage of the link by the enzyme releasing the drug from GFLG peptide.29, 35 In vitro drug release was examined in the presence or absence of papain and tested through RP-HPLC. Figure 4 shows identical elution times for the PTX standard (Figure 4A, peak: 5.6 min) and the released sample (Figure 4B, peak: 5.6 min), thereby indicating that the product released by the nanoparticles may be the drug, PTX. Moreover, the released product at the 5.6 min peak was collected. Then, ESI-MS was applied into analyzing its molecular weight. The most abundant peaks (m/z = 853.96, 875.95, and 891.92) were assigned as [M+H]+, [M+Na]+ and [M+K]+, respectively (Figure 4D). These results further suggested that the released sample at the 5.6 min peak was the drug PTX (m/z = 853). These findings indicated that the aminolysis reaction catalyzed by papain resulted in release of the original structure, PTX. The concentration of PTX released from the carrier at different time points was tested via RPHPLCs. Rapid drug release was observed in the group incubated with papain, demonstrating enzyme-responsive drug release feature. About 50% of PTX was released in the present of papain after 8 h, and approximately 90% of PTX was released after 24 h. In contrast, significantly less PTX was released in buffer without papain (pH = 5.4 or 7.4) after 48 h incubation (Figure 4C). For the sample in buffer without papain (pH 7.4), around 7% of PTX


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was detected to be released after 24 h incubation, and 10% of PTX was released from the nanoparticles after 48 h incubation. Similarly in buffer without papain (pH = 5.4), only 2% and 3% of PTX were released after 24 h and 48 h, respectively. Fetal bovine serum in PBS (pH = 7.4) was chosen as a model for the evaluation of drug release activity of nanoparticles circulating in the bloodstream. In PBS with 10% FBS, 12% and 21% of PTX were released after 24 h and 48 h incubation, respectively. These results indicated that PEGylated dendrimer-GFLF-PTX exhibited high papain selectivity and high stability. Although the hydrolysis of ester bonds of the PTX molecule was unavoidable, the conjugate-based nanoparticles showed high stability, and the vehicle effectively protected PTX during circulation. The lower rate of opsonization, high stability, and minimal drug release during the circulation may contribute to the reduction in side effects and minimal loss in antitumor efficacy. This study has shown that PEG-protected nanoparticles remain stable in circulation, and the enzyme-sensitive nanoparticles could release the hydrophobic drug PTX in the presence of cathepsin B. DLS and SEM were used to evaluate the size and morphology of the particles under different pH conditions with or without enzyme in real-time. The release of the hydrophobic unit leads to a reduction in its self-assembly, which in turn results in less compact nanoparticles that would collapse upon aggregation.40 The size of the particles in buffer (pH 7.4 or 5.4) without papain remained around 60–140 nm for 24 h (Figure S3), indicating no obvious structural collapse. In contrast, the particles incubated with papain were detected as loose, with large and wide PDIs as measured by DLS (Figure 5). Furthermore, morphological assessment via SEM showed similar results. An increase in size and the collapse of particles were observed after 4 h of incubation. These results indicated that the particles could be stable during circulation. The changes in size and morphology, which are governed by cathepsin B, further demonstrated that


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the enzyme-sensitive property and the enzyme-induced collapse of particles contribute to rapid drug release. 3.4. In Vitro Cell Studies The cellular uptake of PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles into 4T1 cells was observed using CLSM. PEGylated dendrimer-GFLF-PTX was modified with Cy5.5 via covalent bonding to track its movement in vitro. Cells were incubated with RPMI1640 containing Cy5.5-labeled nanoparticles (concentration: 20 µg/mL), further treated for CLSM studies. Endocytosis was depicted by red fluorescence that was emitted by the nanoparticles. The intracellular red fluorescence was observed at 2 h post-incubation, whereas the merged yellow fluorescence indicated that the nanoparticles were internalized by the cancer cells and stored in lysosomes (Figure 6A). Similar to the findings of previous reports, PEG, which reduces nonspecific cellular uptake by the MPS and modulates the circulation half-life of nanoparticles, was utilized in the preparation of a “stealthy” nanoparticle. However, high surface density of PEG hinders its cellular uptake.3 Thus, relatively low surface PEG density was chosen to protect the nanoparticles during circulation and to facilitate their relatively rapid cellular internalization after tumor accumulation. These results suggest that the prepared prodrug could be stored in the lysosomes of cancer cells and that PTX is released due to the cleavage of the GFLG-PTX linker by enzyme, ultimately inducing cytotoxicity. The in vitro cytotoxicity of nanoparticles was studied via a cell viability assay on 4T1 cell lines. The cells were incubated with different formulations (PTX and prodrug-based nanoparticles) for 48 h. The CCK-8 assay results demonstrated that nanoparticles did not induce significant cytotoxicity against cancer or normal cells, as shown in Figure S4. In contrast, as shown in Table 1, PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles induced


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marked cytotoxicity to 4T1 cells, which was close to that of free drug PTX, as the very closed half maximal inhibitory concentration (IC50) values (0.67 µg/mL for nanoparticles and 0.52 µg/mL for free PTX). In view of the results of drug release (Figure 4C), upon cellular internalization of the prodrugs (Figure 6A), PTX was released in the presence of cathepsin B. The observed in vitro cytotoxicity further suggested the fast intracellular PTX release from the prodrug-based nanoparticles. Moreover, 3T3 and C2C12 cells were selected as comparative models, as the cathepsin B level in normal cells is much lower than that in tumor cells. Through IC50 calculations shown in Table 1, compared with free PTX, the nanoparticles have much lower cytotoxicity in both 3T3 and C2C12 cells, whereas PTX and nanoparticles showed similar cytotoxicity effects in 4T1 cancer cells. These results may be due to the stability of the nanoparticles in the absence of the enzyme (Figure S3) and the lack of PTX that was released by nanoparticles in normal cells. In another aspect, these results indicated that nanoparticles would be “stealthy” enough to reduce the side effects as they enter normal organs. Furthermore, the capacity of PTX and PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles was evaluated in terms of tumor cell apoptosis. The efficacy of a drug in tumor cells is associated with the suppression of tumor cell growth and the induction of cell apoptosis.41 Through apoptosis analysis, free PTX and the conjugate-based nanoparticles were found to effectively trigger the apoptosis of 4T1 cells. Meanwhile, a similar cell apoptosis ratio of nanoparticles was observed using free PTX (Figure 6B). These results were in agreement with the cytotoxicity findings of the IC50 assay and may be attributed to the relatively rapid cellular uptake and intracellular drug release from the conjugate-based nanoparticles. 3.5. Protein Adsorption Analysis


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PEG was introduced to enhance the solubility and plasma stability of proteins, as well as to reduce immunogenicity.3, 18-19 Thus, we evaluated the interaction of nanoparticles with proteins, using BSA as a model plasma protein. Figure 7 shows that at physiological conditions (pH 7.4), the nanoparticles underwent negligible protein adsorption after 2 h incubation. Moreover, after 36 h under the same conditions, the prodrug-based nanoparticles continued to show minimal protein adsorption, while the free PTX injection showed strong protein adsorption under the same conditions. Thus, the nanoparticles show reduced non-specific protein adsorption compared to free PTX, thereby indicating prolonged blood circulation for nanoparticles. The use of nanoparticles is one of the most effective methods for reducing the rate of protein opsonization by altering the hydrophilicity or neutralizing the surface of the vehicle to avoid sequestration or clearance.19 Opsonization begins immediately after the nanoparticles are introduced into plasma. Therefore, our PEGylated polymer nanoparticles are a promising drug delivery system with improved stability in blood. 3.6. Fluorescence Imaging Analysis Fluorescence imaging is an effective method of monitoring the in vivo distribution and accumulation of a drug in a tumor. The present study utilized this method to monitor the distribution of the prodrug-based nanoparticle through the in-vivo and ex-vivo imaging of normal organs and tumors.42 Figure 8A shows an in vivo image of nanoparticles that have accumulated in a tumor at 6 h after intravenous injection, whereas fluorescence signals are detected in a tumor by using ex vivo imaging. Furthermore, signals were detected in tumors both in-vivo and ex-vivo at 6 h and 12 h (Figure 8A–C). In Figure 8C and 8D, a peak in the fluorescence signals in tumors was observed at 24 h. These results indicated that nanoparticles accumulated in the tumor, released the drug, and were then degraded and cleared out of the body. After tumor accumulation


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(Figure 8), the nanoparticles interacted with the tumor cells and were internalized (Figure 6), which was subsequently followed by the release of the drug (Figure 4). The fluorescence marker Cy5.5 was cleaved from the nanoparticles and cleared out of the body, thus resulting in weaker fluorescence signals. The average intensity of the signals further confirmed the results of in-vivo and ex-vivo imaging (Figure 8D). Owing to the high accumulation into tumors and high stability, the enhanced anticancer efficacy may be mediated by the prodrug-based nanoparticles compared to the free drug. 3.7. In Vivo Antitumor Efficiency The in vivo anti-cancer efficiency and side effects of PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles were measured in mice inoculated with 4T1 cancer cells relative to that using free PTX. The behaviors and body weights of mice as well as the tumor volumes were measured and monitored every other day. Figure 9A shows the time-related increase in tumor volume, and the tumors in all drug and nanoparticles treatment groups resulted in significant growth retardation. The nanoparticles significantly inhibited tumor growth compared to that of the groups treated with PTX and saline. Figure 9 shows that the relative tumor volumes at the 19th day were 972 ± 134% (control group), 902 ± 152% (PTX group), and 383 ± 71% (NPs group), respectively. In particular, after 19 days of therapy, as presented in curves of relative tumor volume (Figure 9A), visual solid tumor size (Figure 9C) and the image of all the tumors (Figure S5), the nanoparticle-treated group showed significantly enhanced antitumor efficiency than the PTX-treated group using this route of administration. The tumor growth and metastatic spread of 4T1 cells in BALB/c mice very closely mimic human breast cancer and this tumor is an animal model for stage IV human breast cancer, thus it can grow very fast without effective treatment. That may be one of reasons that the free drug PTX resulted in low cancer efficacy.


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However, the tumors in the group treated with nanoparticles were significantly smaller than those of the other treatment groups. After treatment, tumor growth inhibition (TGI) and tumor weight were measured and calculated. Figure 9D and 9E indicate that the PTX injection treatment group and the group treated with prodrug based nanoparticles had tumor weights of 994.2 ± 158.1 mg and 465.3 ± 84.8 mg (p < 0.001 vs. PTX injection and p < 0.0001 vs. control), respectively. Furthermore, respective TGIs of 16.6% and 61.0% were observed, indicating that nanoparticles have significantly higher antitumor efficiency. These results could be attributed to the overall enhanced efficacy (SAIR) that was designed. The efforts may due to its negatively charged surface (Figure 3C), appropriate nanoscale size (Figure 3A), and lower rate of opsonization (Figure 7), thereby improving drug stability while in circulation. These features have resulted in its rapid accumulation in tumors via the EPR effect (Figure 8), cell uptake (Figure 6A), and intralysosomal PTX liberation after endocytosis (Figure 4C). In vivo antitumor studies have indicated that the PEGylated dendritic prodrug-based nanoparticles meet the standards of SAIR, thereby improving its overall antitumor efficacy. To evaluate the risk for systemic toxicity due to its injection, the body weights of the mice were monitored. The mice treated with nanoparticles were not observed to have weight loss over the treatment period (Figure 9B), thus indicating no detectable systemic toxicity due to the PTX formulations. In terms of the free PTX injection, no toxicity was observed as indicated by a loss in body weight, and thus the biosafety of nanoparticles was further investigated using the subsequent experiments. 3.8. Immunohistochemical Studies To further examine the therapeutic effect, we detected the apoptotic and proliferation pathway in tumors. As shown in Figure 10B, approximately 61% of TUNEL-positive cells was detected


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in tumors treated with prodrug-based nanoparticles, while obviously lower TUNEL-positive cells (~36%) was calculated in the group of PTX injection. These results suggested that PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles could efficiently induce the apoptosis compared to clinical drug PTX. Ki-67 is a commonly used proliferation marker and was used to further evaluate tumor proliferation. 10, 43-45 The proportion of Ki-67 positive cells detected in the group treated with nanoparticles was less than that treated with clinical drug PTX injection (p < 0.0001). Thereby, a higher antitumor activity in the group of nanoparticles was observed than PTX injection, indicating the rate of proliferation in the group of nanoparticles was significantly lower. Angiogenesis usually plays a critical role during tumor growth, invasion, and metastasis.46 Herein, we evaluated angiogenesis in tumors through detection of the angiogenesis marker CD31. Figure 10 shows the results of optical density analysis, wherein the average MVD of the nanoparticle-treated mice was much lower than that of the two other group samples (p < 0.001), indicating more effective in inhibiting the process of angiogenesis. The above results indicated that the prodrug-based nanoparticles could inhibit the proliferation and promote apoptosis of tumor cells. What is more, the prodrug-based nanoparticles showed preferable property in inhibiting the process of angiogenesis. These findings indicated that Janus PEGylated dendrimer-GFLG-PTX prodrug-based nanoparticles could be employed as efficient vehicles for cancer therapy. First, the prodrug-based nanoparticles contain a negatively charged surface, which promoted its stability during circulation. Second, the PEGylated dendrimer improved the stability of PTX during its transportation. Third, the drug PTX can be rapidly released from the polymeric nanoparticles, as it was attached to the carriers via an enzymesensitive GFLG linker. Finally, since tumor growth is correlated with the deregulation of cell


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proliferation and apoptosis, rapid intracellular drug release may result in significant suppression of tumor growth via induction of tumor cell apoptosis as well as inhibition of tumor cell proliferation. 3.9. Hematological Analysis The biosafety of nanoscale drug delivery system is another concern in the design of novel drug vehicles.47 The first physiological system that a dendrimer prodrug interacts with is blood. It is therefore essential to investigate whether the injection or its constituents would induce toxicity upon administration. PTX injection was reported to induce hematologic toxicity after i.v. injection of a specific dose (dose-limited toxicity).48 Therefore, we designed nanoparticles that could remain in circulation yet be non-toxic to the recipient. Three groups of healthy female mice were treated daily for 10 times as shown in the experimental section. Throughout the course of the therapeutic efficacy studies, repeated injections of nanoparticles were generally well tolerated with no obvious body weight lost observed (Figure S6). At the end of the experiment, the mice were sacrificed and the blood was collected for routine analysis. To further assess the biocompatibility of PTX injection and PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles to blood, hematology and blood routine examination were performed.48 The hematology marker values were mostly within the normal ranges (Figure 11). Meanwhile, the neutrophil granulocyte (GR#) and platelet count (PLT) of mice treated with PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles were similar to that of mice treated with saline. In contrast, those markers in mice treated with clinical agent PTX showed a significant reduction. The observed lower or minimal hematologic toxicity (PTX dose-limited toxicity) might have contributed to the dose improvement. These results indicated that our PEGylated dendrimer-GFLF-PTX prodrug administered as an injection could be a potential drug


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delivery system with high biocompatibility and low hematotoxicity. 4. CONCLUSION In summary, we have successfully designed and prepared a nanoscale system that meets all SAIR requirements for a biocompatible and efficient drug delivery system. The low rate of protein adsorption and minimal to negligible drug release in physiological conditions indicate that the nanoparticles can be “steady” and stable during the circulation. The drug was attached to a carrier using an enzyme-responsive linker and the resultant Janus PEGylated dendrimer-PTX prodrug was optimized via PEGylation. Fluorescence in-vivo and ex-vivo imaging indicated that the nanoparticles easily and rapidly accumulated in the tumor. The drug release studies showed that PTX could be released in a highly efficient manner from the prodrug-based nanoparticles in the presence of an enzyme, while retaining its original structure. The in vitro assays have demonstrated that the nanoparticles could efficiently induce the apoptosis of breast tumor cells, yet elicit a much lower cytotoxic effect on normal cells compared to that using free PTX. By combining the features of the Janus peptide dendrimer, PEG modification, nanoscale system, and enzyme-sensitive linker, the prodrug-based nanoparticles present significantly enhanced antitumor therapeutic indices and biosafety compared to the PTX injection in vivo. The Janus PEGylated dendrimer-GFLF-PTX prodrug in the present study may serve as an efficient and safe nano-vehicle for cancer treatment. AUTHOR INFORMATION Supporting Information The characterization and cytotoxicity of the drug free dendritic polymer, the change of particles incubated with different conditions and the biosafety of nanoparticles are supplied as


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Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org Corresponding Author *

Tel.: +86 28 85414308; fax: +86 28 85410653. E–mail addresses (Luo): [email protected]

*

Tel.:

+86

28

85410336;

fax:

+86

28

85410653.

E–mail

addresses

(Gu):

[email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by International Science and Technology Cooperation Program of China (2015DFE52780), National Natural Science Foundation of China (81361140343, 51373104, 51673127 and 81621003) and Joint Sino-German Research Project of China (GZ905). REFERENCES (1) Sun, Q.; Sun, X.; Ma, X.; Zhou, Z.; Jin, E.; Zhang, B.; Shen, Y.; Van Kirk, E. A.; Murdoch, W. J.; Lott, J. R.; Lodge, T. P.; Radosz, M.; Zhao, Y., Integration of Nanoassembly Functions for an Effective Delivery Cascade for Cancer Drugs. Adv. Mater. 2014, 26 (45), 76157621. (2) Chauhan, V. P.; Jain, R. K., Strategies for Advancing Cancer Nanomedicine. Nat. Mater. 2013, 12 (11), 958-962. (3) Du, X.-J.; Wang, J.-L.; Liu, W.-W.; Yang, J.-X.; Sun, C.-Y.; Sun, R.; Li, H.-J.; Shen, S.; Luo, Y.-L.; Ye, X.-D.; Zhu, Y.-H.; Yang, X.-Z.; Wang, J., Regulating the surface poly(ethylene


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Nanoparticle Size in Hemocompatibility. Toxicology 2009, 258 (2–3), 139-147. (43) Lu, R.-M.; Chang, Y.-L.; Chen, M.-S.; Wu, H.-C., Single Chain Anti-c-Met Antibody Conjugated Nanoparticles for in Vivo Tumor-Targeted Imaging and Drug Delivery. Biomaterials 2011, 32 (12), 3265-3274. (44) Shi, C.; Guo, X.; Qu, Q.; Tang, Z.; Wang, Y.; Zhou, S., Actively Targeted Delivery of Anticancer Drug to Tumor Cells by Redox-responsive Star-shaped Micelles. Biomaterials. 2014, 35, 8711-8722. (45) Wang, J.; Yang, G.; Guo, X.; Tang, Z.; Zhong, Z.; Zhou, S., Redox-responsive Polyanhydride Micelles for Cancer Therapy. Biomaterials. 2014, 35, 3080-3090. (46) Carmeliet, P.; Jain, R. K., Angiogenesis in Cancer and Other Diseases. Nature 2000, 407 (6801), 249-257. (47) Hung, C.-C.; Huang, W.-C.; Lin, Y.-W.; Yu, T.-W.; Chen, H.-H.; Lin, S.-C.; Chiang, W.H.; Chiu, H.-C., Active Tumor Permeation and Uptake of Surface Charge-Switchable Theranostic Nanoparticles for Imaging-Guided Photothermal/Chemo Combinatorial Therapy. Theranostics 2016, 6 (3), 302-317. (48) Huang, X.; Li, L.; Liu, T.; Hao, N.; Liu, H.; Chen, D.; Tang, F., The Shape Effect of Mesoporous Silica Nanoparticles on Biodistribution, Clearance, and Biocompatibility in Vivo. ACS Nano 2011, 5 (7), 5390-5399.


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Figures

Figure 1. The structure and synthesis schemas of the Janus dendritic prodrug and the illustration of its self-assembled nanoparticles. The nanoparticles accumulated to tumor after circulation and released the free drug through intracellular enzymolysis after endocytosis.


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Figure 2. The mass spectrum of the PEGylated dendrimer-GFLF-PTX prodrug.


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Figure 3. The size of the PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticle as measured by (A) DLS, (B) AFM, and (C) zeta potential.


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Figure 4. Drug release was measured using HPLC. PTX injection (A) and PEGylated dendrimer-GFLF-PTX (B) incubated with papain were measured via HPLC. The released sample was gathered and characterized using ESI-MS (D). (C) The concentration of the released drug, PTX, was measured at different time points via HPLC by using UV at a wavelength of 227 nm. Data are expressed as the mean ± SD (n = 3).


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Figure 5. Changes in size and morphology of PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles incubated with an enzyme over time, as measured through DLS and SEM. The real-time size of particles incubated in buffer was recorded. Particles were exposed to papain in McIlvaine’s buffer (pH 5.4). Size distributions were recorded based on intensity.


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Figure 6. Cellular uptake and 4T1 cell apoptosis. (A) Cellular uptake of PEGylated dendrimerGFLF-PTX prodrug-based nanoparticles labeled-Cy5.5 (red) after 2 h incubation. A lysosome tracker (green) was utilized to stain cellular lysosomes (× 10 µm). (B) Cell apoptosis as measured by cell cytometry after labeling 4T1 cells with annexin V-FITC.


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80 Protein adsorption ratio (%)

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70 PTX injection

60

NPs

50 40 30 20 10 0 2

6

12 Time (h)

24

36

Figure 7. Protein adsorption of the nanoparticles (NPs) and PTX injection after incubating at 37°C (pH 7.4) for various time intervals. Bovine serum albumin (BSA) was used as standard (mean ± SD, n = 3).


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Figure 8. Fluorescence imaging studies. (A) In-vivo image at different time points of postinjection (n = 3). (A1–A3, B1-B3) represent imaging after 6 h, 24 h, and 36 h post-injection. After sacrifice, the normal organs and tumor tissues of mice were excised for ex-vivo imaging (n = 3). Among (B1–B3), the tissues of control group were on top, while nanoparticle group on bottom. The accumulation of nanoparticles in tumors (C) after different post-injection times is presented. (D) The average signals of ex-vivo organs and tumors in the NPs group were counted.


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Figure 9. In vivo anticancer efficacy mediated by prodrug-based nanoparticles (NPs) and clinical drug PTX (PTX injection) using saline as control in the 4T1 cancer model (n = 7). Nanoparticle treatment resulted in significant tumor growth inhibition (*p < 0.001, vs. control; **p < 0.001, vs. PTX injection) (A). (B) The body weight shift. After 19 days, tumors were imaged (C) and weighed (*p < 0.0001, vs. control; **p < 0.001, vs. PTX injection) (D). (E)Tumor growth inhibition (TGI).


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Figure 10. TUNEL, CD31 and Ki-67 staining of tumors (all tissues: × 400) (A). During the three methods, brown all represent the positive-staining. (B) The ratio of the number of apoptotic cells to the total number of tumor cells (apoptotic index) was calculated (*p < 0.0005 vs. control and **p < 0.01 vs. PTX injection). (C) The ratio of CD31 to the total area of each photograph was calculated as tumor microvessel density (MVD). (D) Ki-67-positive area/Total area was counted as Ki-67 density (*p < 0.0001, vs. control and **p < 0.0001 vs. PTX injection) (mean ± SD, n = 7).


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6 5 4 3 2

109/L) WBC ((10

40

MCV (fL)

30 20 10

1

PTX injection

Control

NPs

160 140 120

MCH (pg)

100 80 60 40

PTX injection

Control

25

1000

20

800

Control

PTX injection

0.4

0 Control

PTX injection

Control

NPs

MCHC (g/L)

300 200

0.1

PTX injection

NPs

2.5

**

2 1.5 1 0

0

Control

NPs

0.5

100

0

PTX injection

3

400

0.2

**

200

500

0.3

NPs

400

10

NPs

PTX injection

600

15

0

0

2

NPs

5

20

4

PLT (109/L)

Control

6

0

0

0

GR#(109/L)

RBC (1012/L)

7

HGB (g/L)

8

50

8

HCT (L/L)

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Control

PTX injection

NPs

Control

PTX injection

NPs

Figure 11. The hematological analysis of mice that received different treatments, including saline, PTX injection and PEGylated dendrimer-GFLF-PTX prodrug-based nanoparticles (NPs). After 19 days of treatment, hematological analysis was performed (n = 7). Hematologic toxicity induced by PTX injection was observed as a decrease in platelet (PLT) and neutrophil granulocyte (GR#) (red frame). In contrast, mice treated with nanoparticles did not exhibit any signs of toxicity (**p < 0.0001 compared to that using a PTX injection).


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Table 1. The IC50 Test of Breast Cancer Cell Line (4T1, Murine) and Normal Cell Lines (3T3 and C2C12, Murine) Treated with PTX Injection and Nanoparticles for 48 h, respectively. Cell lines

IC50 (μg/mL) PTX injection

Nanoparticles

4T1

0.52

0.67

3T3

0.05

0.35

C2C12

0.02

0.06


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Graphical abstract


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Enzyme-Sensitive and Amphiphilic PEGylated Dendrimer-Paclitaxel

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