Biodegradable Micelles Based on Poly(ethylene glycol)-b

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Biodegradable Micelles Based on Poly(ethylene glycol)-b-Polylipopeptide Copolymer: A Robust and Versatile Nanoplatform for Anticancer Drug Delivery Min Qiu, Jia Ouyang, Huanli Sun, Fenghua Meng, Ru Cheng, Jian Zhang, Liang Cheng, Qing Lan, Chao Deng, and Zhiyuan Zhong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10533 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 8, 2017

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ACS Applied Materials & Interfaces

Biodegradable Micelles Based on Poly(ethylene glycol)-b-Polylipopeptide Copolymer: A Robust and Versatile Nanoplatform for Anticancer Drug Delivery

Min Qiu1, Jia Ouyang2, Huanli Sun1, Fenghua Meng1, Ru Cheng1, Jian Zhang1, Liang Cheng1, Qing Lan2, Chao Deng1,*, and Zhiyuan Zhong1,*

1

Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional

Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, China.

2

Department of Neurosurgery, The Second Affiliated Hospital of Soochow University, Suzhou,

215004, China.

ABSTRACT: Poly(ethylene glycol)-b-polypeptide block copolymer micelles with excellent safety are one of the most clinically studied nanocarriers for anticancer drug delivery. Notably, self-assembled nanosystems based on hydrophobic polypeptides showing typically a low drug loading and burst drug release are limited to preclinical studies. Here, we report that poly(ethylene glycol)-b-poly(α-aminopalmitic acid) (PEG-b-PAPA) block copolymer could be easily prepared with tailored Mn through ring-opening polymerization of α-aminopalmitic acid N-carboxyanhydride (APA-NCA). Interestingly, PEG-b-PAPA copolymers exhibited superb solubility in common organic solvents (including CHCl3, CH2Cl2, and THF) while formed stable nanomicelles in phosphate buffer, with a small size of 59 nm and low critical micelle concentration of 2.38 mg/L. These polylipopeptide micelles (Lipep-Ms) allowed

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facile loading of potent anticancer drug, docetaxel (DTX), likely due to existence of strong interaction between lipophilic drug and polylipopeptide in the core. Notably, cRGD peptide-functionalized Lipep-Ms (cRGD-Lipep-Ms) were also obtained with similar biophysical characteristics. The in vitro studies showed efficient cellular uptake of DTX-loaded cRGD-Lipep-Ms by B16F10 cells and fast intracellular drug release due to the enzymatic degradation of PAPA blocks in endo/lysosome, leading to pronounced anticancer effect (IC50 = 0.15 µg DTX equiv./mL). The in vivo therapy studies showed that DTX-cRGD-Lipep-Ms exhibited superior tumor growth inhibition of B16F10 melanoma, improved survival rate and little side effects as compared to free DTX. These polylipopeptide micelles appear as a promising and robust nanoplatform for anticancer drug delivery. KEYWORDS: polypeptide, lipopeptide, micelles, docetaxel, drug delivery, cancer therapy

1. INTRODUCTION Poly(ethylene glycol)-b-polypeptide block copolymer micelles with excellent safety are one of the most clinically studied nanocarriers for anticancer drug delivery.1, 2 For example, paclitaxel

(PTX),

doxorubicin

(DOX),

and

cisplatin-loaded

polypeptide-based

nanotherapeutics have proceeded into phase II and III clinical trials, and achieved enhanced therapeutic efficacy against intractable tumors including triple-negative breast cancer, pancreatic cancer and glioblastoma, and reduced side effects in comparison to free drugs.3-5 Especially, PTX-loaded PEG-b-poly(aspartate-4-phenyl-1-butanolate) micelles (NK105) have exhibited superior therapeutic efficacy on gastric and breast cancers than conventional PTX-Cremophor formulation in phase III clinical trials.3, 6 Cisplatin-loaded PEG-b-poly(Lglutamic acid) (NC6004) nanotherapeutics with ordered α-helical bundles in the core display remarkably enhanced stability and small size (20 nm), and have advanced into phase III clinical evaluation for the treatment of intractable pancreatic cancer in 2013.7, 8 However,

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these nanoformulations mostly based on hydrophilic polypeptides, usually involve multi-step synthesis including protection and deprotection of functional groups and drug conjugation. Hydrophobic polypeptides based on natural hydrophobic amino acids (e.g. leucine, phenylalanine, and methionine)9-12 as well as hydrophobically modified hydrophilic amino acids (e.g. γ-benzyl L-glutamate, β-benzyl L-aspartate, ε-benzyloxycarbonyl-L-lysine)13-17 have also been employed to fabricate micelles and polymersomes for controlled drug delivery. These self-assembled nanosystems are, however, often plagued with low drug loading and burst drug release. Lipid-based nanosystems including liposomes, lipid micelles, solid lipid nanoparticles, and lipid-polymer hybrid nanoparticles that show excellent biocompatibility are among the most important vehicles for controlled delivery of therapeutic agents.18-21 For example, liposomal formulations of doxorubicin, daunorubicin, and vincristine have been approved for the treatment of Kaposi’s sarcoma, multiple myeloma, acute lymphoid leukemia, ovarian and breast cancers.22, 23 Lipid-polymer hybrid nanoparticles have shown improved drug loading and sustained drug release.24-26 Farokhzad et al. reported that lipid-PLGA nanoparticles achieved significantly improved encapsulation efficiency of docetaxel (DTX) compared to PLGA-PEG and PLGA nanoparticles.27 Here, we designed and prepared a robust and versatile polylipopeptide nanoplatform based on PEG-b-poly(α-aminopalmitic acid) (PEG-b-PAPA) block copolymers for anticancer drug delivery (Scheme 1). PEG-b-PAPA copolymers with tailored Mn were easily prepared through ring-opening polymerization of α-aminopalmitic acid N-carboxyanhydride (APA-NCA). Interestingly, PEG-b-PAPA copolymers exhibit superb solubility in common organic solvents (e.g. CHCl3, CH2Cl2, and THF) and form stable polylipopeptide micelles (Lipep-Ms) that show efficient loading and intracellular triggered release of potent anticancer drugs like DTX. Lipopeptides with excellent biocompatibility were reported to be promising

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adjuvants on developing anticancer vaccines,28 and could self-assemble into nanofibers for the fabrication of bone-mimetic materials or encapsulation of hydrophobic chemotherapeutics like camptothecin to treat various cancers.29-31 Our results show that DTX-loaded cRGD peptide-functionalized polylipopeptide micelles exhibit superior tumor growth inhibition of B16F10 melanoma, improved survival rate and little side effects as compared to free DTX.

Scheme 1. Formation, efficient DTX encapsulation and intracellular triggered drug release of robust polylipopeptide micelles based on poly(ethylene glycol)‑b‑poly(α-aminopalmitic acid) (PEG-PAPA) block copolymer.

2. EXPERIMENTAL SECTION 2.1. Synthesis of α-Aminopalmitic Acid N-Carboxyanhydride (APA-NCA) Monomer. Under a nitrogen atmosphere, α-pinene (2.7 mL, 16.5 mmol) and triphosgene (1.64 g, 5.5 mmol) were added to a solution of α-aminopalmitic acid (3.0 g, 11.1 mmol) in dry THF (80 mL). After stirring at 50 °C for about 1 h, the reaction mixture was concentrated, and the residue was precipitated in petroleum ether to give crude APA-NCA. The crude product was redissolved in THF, dried with anhydrous MgSO4, and recrystallized at least twice from THF/petroleum ether to obtain APA-NCA (1.0 g, 45.5%). 1H NMR (400 MHz, DMSO-d6, Figure 1A, δ): 9.07 (s, 1 H, -CHNHCO-), 4.41 (m, 1 H, -COCHNH-), 1.65 (m, 2 H, -CH2CH2CH-), 1.29 (m, 24 H, -CH2(CH2)12CH3), 0.84 (t, 3 H, -CH2CH3).

4


13

C NMR (100

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MHz, DMSO-d6, Figure S1, δ): 171.66, 151.96, 57.04, 31.33, 30.95, 29.06-28.43, 24.26, 22.12, 13.93. Elem. Anal. Calcd for C17H31NO3: C, 68.65; H, 10.51; N, 4.71. Found: C, 68.37; H, 10.07; N, 4.65. Electrospray ionization mass spectrometry (ESI-MS, m/z): [M+Na]+ calcd. for C17H21NO3 320.23; found 320.22.

2.2. Synthesis of mPEG-b-PAPA Polylipopeptides. mPEG-b-PAPA polylipopeptides were synthesized through the ring opening polymerization of APA-NCA using mPEG-NH2 as a macroinitiator. Typically, to a solution of APA-NCA (0.19 g, 0.64 mmol) in DMF was quickly added a solution of mPEG5k-NH2 (0.2 g, 0.04 mmol) in DMF under a nitrogen atmosphere. The reaction mixture was stirred for 72 h at 35 oC. The resulting mPEG-b-PAPA copolymer was precipitated in excess diethyl ether, further purified by re-dissolving in dichloromethane and precipitating in diethyl ether for three times, and dried in vacuo. Yield: 91%. 1H NMR (600 MHz, CDCl3/CF3COOH (9/1, v/v), Figure 1B, δ): 4.62 (1 H, -HNCH(CH2)CO-), 3.85 (4 H, -OCH2CH2O-), 3.52 (3 H, CH3O-), 1.76 (2 H, -CH(NH)CH2CH2-),

1.27

(24

H,

-CH2(CH2)12CH3),

0.88

(3

H,

-CH2CH3).

Acrylate-PEG-b-PAPA (AA-PEG-b-PAPA) was synthesized in a similar way except that AA-PEG-NH2 with an Mn of 6.0 kg/mol was used as a macroinitiator. Yield: 83%. 1H NMR (600 MHz, CDCl3/CF3COOH (9/1, v/v), Figure S2, δ): AA moieties: 5.85, 5.30, PEG: 3.76; PAPA moieties: 4.52, 1.86, 1.24, 0.87. c(RGDfC) (cRGD) was conjugated onto AA-PEG-b-PAPA through the thiol-ene click reaction in DMF with UV irradiation (320-390 nm, 50 mW/cm2) for 10 min in the presence of I2959 photo-initiator. cRGD-PEG-b-PAPA was isolated through dialysis against DMF for 24 h and then deionized water for 48 h (MWCO 7000 Da) followed by lyophilization. Yield: 90%. 1H NMR (600 MHz, CF3COOD, Figure S3, δ): cRGD: 7.25, PEG: 3.85; PAPA: 4.23, 1.75, 1.27, 0.87. The degree of cRGD conjugation was determined to be 98% by measuring the arginine content using

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9,10-phenanthrene-quinone method according to previous report.32

2.3. Micelle Formation. Lipep-Ms were prepared by dropwise addition of phosphate buffer (PBS, 10 mM, pH 7.4) to a DMF solution of mPEG-b-PAPA under stirring at r.t., followed by extensive dialysis (Spectra/Pore, MWCO 7000 Da) against PBS for 8 h. cRGD-functionalized PEG-b-PAPA polylipopeptide micelles (cRGD-Lipep-Ms) were prepared in a similar way except that a DMF solution of cRGD-PEG-b-PAPA and mPEG-b-PAPA (2/8, mol/mol) was employed. To monitor the cellular uptake and intracellular

trafficking

behaviors,

near-infra-red

fluorescence

probe

Cy5-labeled

polylipopeptide micelles (Cy5-Lipep-Ms and Cy5-cRGD-Lipep-Ms) were obtained by incorporating PEG-b-PAPA-Cy5 conjugate that was prepared by reacting terminal amino groups in PEG-b-PAPA with NHS-activated Cy5.

2.4. Encapsulation and In Vitro Release of DTX. DTX-loaded Lipep-Ms (DTX-Lipep-Ms) were prepared by dropwise addition of PBS (4 mL) to a mixture of mPEG-b-PAPA copolymer (1 mL, 5 mg/mL) and DTX (88.5 µL, 10 mg/mL) in DMF under stirring at r.t. followed by dialysis (MWCO 7000) against PBS for 8 h. The dialysis medium was

refreshed

every

2

h.

In

a

similar

way,

DTX-loaded

cRGD-Lipep-Ms

(DTX-cRGD-Lipep-Ms) were prepared by addition of PBS to a mixture of mPEG-b-PAPA, cRGD-PEG-b-PAPA, and DTX in DMF. The drug loaded in the micelles was extracted using DMF, and the amount of DTX was measured using HPLC (Waters 1525) with UV detection at 243 nm. A mixture of acetonitrile and water (6/4, v/v) was employed as the mobile phase. Drug loading content (DLC) and drug loading efficiency (DLE) of micelles were determined using the following formulas: DLC (wt.%) = (weight of loaded drug/total weight of polymer and loaded drug)×100

6


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DLE (%) = (weight of loaded drug/weight of drug in feed) ×100 The release behavior of DTX from the micelles was studied in phosphate buffered saline (PBS, pH 7.4) with 1% tween 80 at 37 oC either in the presence or absence of 10% FBS. Tween 80 was used to improve the solubility of DTX in PBS. Briefly, DTX-loaded micelle solution (0.6 mL) was transferred to a dialysis tube (MWCO 12000-14000), which was immediately immersed in 25 mL of PBS with 1% tween 80 at 37 oC. At predetermined time intervals, 5 mL of release media was taken out and refilled with an equal volume of fresh media. The amount of DTX released from micelles was determined by HPLC as described above. The release experiments were conducted in triplicate. The results presented are mean ± standard deviations (SD).

2.5. In Vivo Antitumor Efficacy of DTX-loaded Micelles. In vivo antitumor activity of DTX-loaded micelles was evaluated in subcutaneous B16F10 melanoma-bearing mice. Treatment was started when the tumor reached a volume of about 50 mm3, and this day was designated as day 0. Melanoma-bearing mice were randomly divided into six groups (seven mice/group). Four groups were treated with DTX-cRGD-Lipep-Ms, DTX-Lipep-Ms, free DTX (10 mg DTX equiv./kg) and PBS, respectively. The drug was given on day 0, 2, 4, and 6. The rest two groups were treated with DTX-cRGD-Lipep-Ms at a single dose of 40 or 80 mg DTX equiv./kg. The tumor growth and body weight were monitored every two days. The tumor volume was calculated by the formula: V = L× W2/2 (L and W are the length and width of tumors, respectively). The relative tumor volume was normalized by the initial tumor volume.

3. RESULTS AND DISCUSSION

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3.1. Synthesis and Characterization of mPEG-b-PAPA and cRGD-PEG-b-PAPA Block Copolymers. mPEG-b-PAPA block copolymers were readily obtained through direct polymerization of APA-NCA that was synthesized by cyclization of α-aminopalmitic acid in THF using triphosgene (Scheme 2). α-Pinene was employed as a hydrochloride scavenger to prevent byproduct formation during NCA synthesis.33-35 1H NMR of APA-NCA displayed clear signals at δ 4.41 and 9.07 owing to methine and amide protons of NCA ring, 1.29 and 1.65 to methylene protons, and 0.89 to methyl protons (Figure 1A).

13

C NMR detected

besides 15 alkane carbons at δ 13.93-57.04 also two carbonyl carbons at δ 151.96 and 171.66, respectively (Figure S1). ESI-MS data displayed a mass of 302.2, corresponding to the exact mass of APA-NCA plus sodium cation. The successful synthesis of APA-NCA was further validated by elemental analysis that showed a composition close to that calculated. The polymerization of APA-NCA using mPEG-NH2 (Mn = 5.0 kg/mol) as a macroinitiator furnished mPEG-b-PAPA in high yields. The characteristic signals of PEG (δ 3.85 and 3.52) and PAPA (δ 0.88, 1.27 and 1.76) were clearly observed in 1H NMR spectrum (Figure 1B) of mPEG-b-PAPA (Table 1, entry 2). The Mn of mPEG-b-PAPA block copolymers calculated by comparing the intensity of signals at δ 0.88 (methyl protons of PAPA) to δ 3.85 (methylene protons of PEG) was close to the design (Table 1). Hence, the Mn of mPEG-b-PAPA could be easily adjusted by varying APA-NCA/mPEG-NH2 feed ratios. Notably, MALDI-TOF showed that mPEG-b-PAPA had a narrow distribution with an Mn in close proximity to that determined by 1H NMR (Figure 1C). As shown in Table 1, GPC further confirmed that mPEG-b-PAPA copolymers had tailored molecular weights and narrow distribution (Mw/Mn=1.03-1.21). Interestingly, PAPA homopolymers and mPEG-b-PAPA copolymers exhibited excellent solubility in common organic solvents like CHCl3, CH2Cl2, and THF (Table S1). In a similar way, polymerization of APA-NCA initiated by heterobifunctional AA-PEG-NH2 (Mn = 6.0 kg/mol) yielded AA-PEG-b-PAPA copolymer

8


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(Scheme 2). 1H NMR and GPC measurements showed that AA-PEG-b-PAPA had a prescribed Mn and a narrow distribution (Mw/Mn = 1.23) (Figure S2, Table 1). AA-PEG-b-PAPA (Table 1, entry 6) underwent thiol-ene click reaction with c(RGDfC) to afford cRGD-PEG-b-PAPA (Scheme S1). Thiol-ene click reaction has been employed for the efficient conjugation of thiol-containing small molecules to alkene-functionalized polymers.36, 37 1

H NMR spectrum of cRGD-PEG-b-PAPA showed besides signals of PEG and PAPA also

resonances of cRGD at δ 7.25 (Figure S3). The degree of cRGD conjugation, determined by measuring the arginine

content using 9,10-phenanthrene-quinone reagent through

fluorometric assay32 and based on calibration curve (Figure S4), was about 98%. The enzymatic degradation of mPEG-b-PAPA was studied in vitro and in vivo. Figure S5 showed that mPEG-b-PAPA polymer films while kept intact in Tris-HCl buffer within 10 d displayed a cracked morphology in the presence of proteinase K, indicating that PAPA is enzymatically degradable. The in vivo degradation experiments demonstrated a gradual degradation of mPEG-b-PAPA polymer discs following subcutaneous implantation in the back of mice (Figure S6A). Notably, polymer discs were completely degraded in 21 days. It should further be noted that the subcutaneous implantation of mPEG-b-PAPA discs caused mild inflammatory response on day 10 and practically no inflammation on day 21 (Figure S6B), indicating good biocompatibility of mPEG-b-PAPA polymer as well as its degradation products.

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Scheme 2. Synthesis of mPEG-b-PAPA and AA-PEG-b-PAPA block copolymers. Conditions: (i) triphosgene, α-pinene, THF, 50 oC, 1 h; (ii) mPEG-NH2 or AA-PEG-NH2, DMF, 35 oC, 72 h.

10


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Figure 1. (A) 1H NMR spectrum of APA-NCA in DMSO-d6 (400 MHz). (B) 1H NMR spectrum of mPEG-b-PAPA (Table 1, entry 2) in CDCl3/CF3COOH (9/1, v/v) (600 MHz). (C) MALDI-TOF spectrum of mPEG-b-PAPA (Table 1, entry 2).

Table 1. Synthesis of diblock copolylipopeptidesa

11



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Mw/Mnb

Mn (kg/mol) entry

copolymers

c

yield

H NMRb

GPCc

GPC

8.0

8.0

9.7

1.03

88

2

9.0

8.5

10.8

1.14

91

3

11.0

10.9

11.4

1.03

86

4

13.0

12.5

14.6

1.03

84

5

16.0

14.8

18.6

1.21

94

10.0

10.2

12.7

1.23

83

1

design 1

6 a

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mPEG-b-PAPA

AA-PEG-b-PAPA

(%)

mPEG-b-PAPA and AA-PEG-b-PAPA were synthesized by using mPEG5k-NH2 and AA-PEG6k-NH2

as macro-initiator, respectively. bCalculated from 1H NMR. cDetermined by GPC (eluent: DMF (entries 1-4 and 6) or CHCl3 (entry 5); flow rate: 0.8 mL/min; 40 oC; standard: poly(methyl methacrylate)).

3.2. Micelle Formation, Loading of Docetaxel, and In Vitro Drug Release. Polypeptides and hybrids with excellent biocompatibility, biodegradability, versatility, and unique hierarchical structure, have attracted much attention in the fields of controlled drug delivery.38-44 Here, mPEG-b-PAPA with an Mn of 8.5 kg/mol (Table 1, entry 2) was chosen to prepare micelles (Lipep-Ms) by solvent exchange method. The resulting micelles exhibited a unimodal distribution and small size of about 57 nm (Table 2). Recent studies disclosed that nanomedicines with a diameter of around 50 nm had superior tumor retention and therapeutic efficacy.45, 46 CD spectrum of Lipep-Ms showed a negative peak at 200 nm and positive peak at 220 nm (Figure S7), indicating that PAPA blocks in micellar core adopt a random coil conformation. The critical micelle concentrations (CMC) determined using pyrene as a fluorescence probe was approximately 2.38 mg/L (Table 2). Cryo-TEM micrograph demonstrated that Lipep-Ms had a spherical morphology, and a small size close to that observed by DLS (Figure 2A). Notably, Lipep-Ms exhibited decent stability with little size

12


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change under physiological conditions (pH 7.4, 37 oC) for over one month, and against 10% FBS at 37 oC for over 8 h (Figure S8). The relatively low CMC and high colloidal stability of Lipep-Ms could be attributed to the strong hydrophobic interactions of the lipid groups.47, 48 Similarly,

cRGD-Lipep-Ms

were

readily

prepared

from

mPEG-b-PAPA

and

cRGD-PEG-b-PAPA. The PEG block of cRGD-PEG-b-PAPA was designed to be longer than that of mPEG-b-PAPA (6.0 vs 5.0 kg/mol) to facilitate the exposure of cRGD ligand on micelle surface. The molar ratio of mPEG-b-PAPA and cRGD-PEG-b-PAPA was fixed at 4:1. We found that 20% cRGD surface density had optimal tumor-targetability toward αvβ3 receptor-overexpressing U87MG and B16 cancer cells.49,

50

Table 2 shows that

cRGD-Lipep-Ms had comparable size, PDI, Zeta potential and CMC to those of Lipep-Ms.

Figure 2. Characterization and in vitro cytotoxicity of polylipopeptide micelles (Lipep-Ms) and

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cRGD-decorated Lipep-Ms (cRGD-Lipep-Ms). (A) The size and size distribution of Lipep-Ms measured by DLS and Cryo-TEM. (B) In vitro DTX release from DTX-cRGD-Lipep-Ms in PBS or PBS containing 10 % FBS. Data are presented as mean ± SD (n = 3). (C) In vitro DTX release from DTX-cRGD-Lipep-Ms in B16F10 cells. The release time was counted as from the point adding DTX-cRGD-Lipep-Ms into the cells. Data are presented as mean ± SD (n = 3). (D) Flow cytometry of B16F10 cells following 4 h incubation with Cy5-cRGD-Lipep-Ms and Cy5-Lipep-Ms (3 µM Cy5). Cells treated with PBS were used as a control. (E) CLSM images of B16F10 cells following 4 h incubation with Cy5-cRGD-Lipep-Ms and Cy5-Lipep-Ms. The scale bars correspond to 20 µm. (F) Antitumor activity of DTX-cRGD-Lipep-Ms, DTX-Lipep-Ms and free DTX against B16F10 cells determined by MTT assays. (G) Immunofluorescene showing microtubule of B16F10 cells following treatment with DTX-cRGD-Lipep-Ms, DTX-Lipep-Ms, Free DTX and PBS (6 µg DTX equiv./mL). Green and blue staining are anti-β3 tubulin antibody and DAPI, respectively.

Table 2. Characterization of blank micelles

a

a

a

zetab

CMCc

(mV)

(mg/L)

entry

micelles

size (nm)

PDI

1

Lipep-Ms

59 ± 1.2

0.17

-1.23±0.64

2.38

2

cRGD-Lipep-Ms

58 ± 0.8

0.18

-1.88±0.76

2.26

Determined by DLS (n = 3). bMeasured by electrophoresis at 25 oC in PBS (n = 3). cDetermined by

fluorescence measurement.

DTX, an anti-mitotic chemotherapeutics, has been approved by FDA for the treatment of breast cancer, gastric cancer, prostate cancer, lung cancer, melanoma, and so on.51, 52 The loading of DTX showed that at a theoretical drug loading content (DLC) of 15 wt.%, Lipep-Ms had a high DLC of 11.1 wt.% and drug loading efficiency (DLE) of 70.8% (Table 3). DTX nanomedicines are mostly reported to have a low drug loading level (< 8 wt.%).53-55 The high drug loading of Lipep-Ms is likely attributed to the enhanced interaction between polylipopeptide and DTX.47, 48 Moreover, DTX-loaded Lipep-Ms revealed a small size of 59

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nm. Similarly, cRGD-Lipep-Ms exhibited a high DLC of 11.6 wt.% and DLE of 74.3%. Figure 2B showed that less than 30% drug was released from cRGD-Lipep-Ms under physiological conditions (pH 7.4, 37 oC) in 24 h either with or without 10% FBS, confirming that DTX-cRGD-Lipep-Ms has good stability. We then investigated the drug release behavior of DTX-cRGD-Lipep-Ms in B16F10 tumor cells. Interestingly, the results showed that ca. 45% and 60% of DTX was released in B16F10 cells in 4 and 8 h, respectively (Figure 2C). The micelles based on PEG-b-PAPA have, therefore, desired features of high DTX loading, good stability, small size, and fast intracellular drug release.

Table 3. Characterization of DTX-Lipep-Ms and DTX-cRGD-Lipep-Ms in PBS (theoretical DLC = 15 wt.%) DLC

DLE

Zeta

(wt.%)b

(%)b

(mV)c

0.17

11.1

70.8

-1.14±0.64

0.18

11.6

74.3

-2.78±1.55

Size

a

Micelles

(nm)a

DTX-Lipep-Ms

59±0.4

DTX-cRGD-Lipep-Ms

58±1.6

PDIa

Determined by DLS. b Determined by HPLC. c Measured by electrophoresis at 25 oC in PBS.

3.3. Cellular Uptake and In Vitro Antitumor Activity of cRGD-Lipep-Ms. MTT assays in normal L929 fibroblast cells and B16F10 cells cancer cells demonstrated that blank cRGD-Lipep-Ms were practically non-cytotoxic at concentrations ranging from 0.1 to 1.0 mg/mL (Figure S9A), and α-aminopalmitic acid did not cause cytotoxicity even at 100 µg/mL (Figure S9B), supporting that both micelles and their degradation products have decent in vitro biocompatibility. cRGD peptide has shown to facilitate the cellular uptake of micelles into αvβ3 integrin-overexpressing cancer cells.56-58 The cellular uptake of Cy5-labeled Lipep-Ms was studied in αvβ3-positive B16F10 melanoma cells by flow cytometry and CLSM. Figure 2D showed clearly efficient internalization of Cy5-cRGD-Lipep-Ms, which gave

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about 2-fold higher cellular Cy5 fluorescence than Cy5-Lipep-Ms. In contrast, no difference was observed in the cellular uptake of Cy5-cRGD-Lipep-Ms and Cy5-Lipep-Ms in αvβ3 negative MCF-7 breast cancer cells (Figure S10). CLSM images displayed a much stronger Cy5 fluorescence in B16F10 cells following 4 h incubation with Cy5-cRGD-Lipep-Ms than with Cy5-Lipep-Ms under otherwise the same conditions (Figure 2E). Notably, DTX-cRGD-Lipep-Ms exhibited a high antitumor activity in B16F10 cells with a half-maximal inhibitory concentration (IC50) of 0.15 µg DTX equiv./mL (Figure 2F), which was comparable to that of free DTX, supporting efficient internalization and fast drug release in B16F10 cells. In comparison, 2.6-fold lower IC50 was observed for the non-targeted DTX-Lipep-Ms (IC50 = 0.39 µg DTX equiv./mL). DTX is known to induce cytotoxic effect by binding to β tubulin subunit.59 The immunofluorescence staining showed clearly that the microtubules

in

B16F10

cells

were

completely

disrupted

by

free

DTX

and

DTX-cRGD-Lipep-Ms (Figure 2G). It is evident that cRGD-Lipep-Ms can efficiently deliver and release DTX to B16F10 cells, affording a high antitumor effect.

3.4. Blood Circulation, Biodistribution and Therapeutic Efficacy. The in vivo pharmacokinetic studies showed that Cy5-cRGD-Lipep-Ms and Cy5-Lipep-Ms possessed a long circulation time with an elimination phase half-life of 2.75 h and 2.33 h, respectively (Figure 3A). Considerable amount of Cy5-cRGD-Lipep-Ms and Cy5-Lipep-Ms was observed even after 24 h post injection. The in vivo biodistribution experiments in B16F10 tumor-bearing C57BL/6 mice revealed a high tumor DTX level of 7.9 %ID/g at 6 h post injection of DTX-cRGD-Lipep-Ms, which was about 2.5 and 5.5-fold higher than that for DTX-Lipep-Ms (2.9 %ID/g) and free DTX (1.3 %ID/g), respectively (Figure 3B). Moreover, DTX-cRGD-Lipep-Ms caused reduced drug accumulation in the heart, liver, spleen and kidney as compared to free DTX. Notably, tolerability studies in C57BL/6 mice showed a

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high

maximum-tolerated

dose

(MTD)

of

over

80

mg

DTX

equiv./kg

for

DTX-cRGD-Lipep-Ms (Figure S11).

Figure 3. (A) In vivo pharmacokinetics of Cy5-cRGD-Lipep-Ms and Cy5-Lipep-Ms in C57BL/6 mice (Cy5 dosage = 6 nmol). Cy5 levels were determined by fluorescence spectroscopy. (B) Quantification of DTX accumulated in different organs and tumors measured by HPLC (*p

Biodegradable Micelles Based on Poly(ethylene glycol)-b

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