Targeting Prostate Carcinoma by G3-C12 Peptide Conjugated

Article pubs.acs.org/molecularpharmaceutics

Targeting Prostate Carcinoma by G3-C12 Peptide Conjugated N‑(2Hydroxypropyl)methacrylamide Copolymers Yang Yang, Lian Li, Zhou Zhou, Qingqing Yang, Chong Liu, and Yuan Huang* Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, People’s Republic of China

ABSTRACT: Prostate carcinoma is the second leading cause of cancer-related deaths. Increased expression of membrane-bound galectin-3 by prostate carcinoma cell has been found to correlate with more poorly differentiated and increased metastatic potential. In the present study, different amount of galectin-3-binding peptide, G3-C12 (the sequence ANTPCGPYTHDCPVKR), was attached to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers as targeting moiety. The results of qPCR and competitive binding test indicated that the expression level of galectin-3 in two metastatic prostate carcinoma cell lines (PC-3 and DU145 cells) could be significantly suppressed by the addition of G3-C12-modified HPMA copolymers (PG1 and PG2), demonstrating the high affinity of PG1 and PG2 to galectin-3. Due to the multivalent effects of moieties, the uptake of copolymers was remarkably enhanced with the increasing amount of conjugated G3-C12 peptide. A higher internalization of PG1 and PG2 occurred in PC-3 cells via caveolin- and clathrin-mediated endocytosis, whereas a clathrin-mediated uptake process was involved in DU145 cells. The in vivo biodistribution and pharmacokinetics of nonmodified (131I-pHPMA) and G3-C12-modified (131I-PG1 and 131I-PG2) copolymers were estimated on a well-established mice model bearing PC-3 xenografts by 131I-SPECTimaging. Higher tumor accumulation of 131I-PG1 (1.60 ± 0.08% ID/g, p < 0.05) and 131I-PG2 (1.54 ± 0.06% ID/g, p < 0.05) was observed compared with 131I-pHPMA (1.19 ± 0.04% ID/g) at 2 h post-intravenous injection. Although the amount of conjugated G3-C12 peptide performed a remarkable in vitro effect on the affinity and internalization of HPMA copolymers to the galectin-3 overexpressed prostate carcinoma cells, the molecular weight and ligand modification all play important roles on their in vivo tumor accumulation. KEYWORDS: galectin-3, G3-C12 peptide, prostate carcinoma, HPMA copolymers

1. INTRODUCTION

Among the variety of polymers frequently applied as drug carriers for cancer therapy, water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers emerged as one of the most promising candidates owing to their unique characteristics such as good compatibility, nonimmunogenicity, nontoxicity, and multifunctionality for the easy conjugation of various drugs and targeting moieties.4,5 The enhanced permeability and retention (EPR) effect is the predominant mechanism by which

Prostate cancer is the most common malignant tumor for males in developed countries and regions. Its mortality rate has been rising up in Asia recently.1 Although androgen ablation, the most common palliative treatment in clinic, could temporarily suppress the growth and progression of prostate cancer, it is only for hormone-refractory prostate cancers.2 With regard to patients who were resistant to metastatic prostate cancers, chemo- and radiotherapies are applied.3 However, the absence of specificity of these strategies may lead to the undesirable biodistribution of therapeutics in normal tissues and induce more side effects. Therefore, tumor-targeting delivery systems are developed to actively and specifically direct anticancer drugs to the metastatic prostate cancer cells. © XXXX American Chemical Society

Special Issue: Recent Molecular Pharmaceutical Development in China Received: January 27, 2014 Revised: June 10, 2014 Accepted: June 23, 2014

A

dx.doi.org/10.1021/mp500083u | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

mechanisms on PC-3 and DU145 cells as well as in vivo biodistribution on mice model bearing PC-3 xenografts were investigated.

soluble macromolecular anticancer drugs exhibit their therapeutic effect on solid tumors.6 It was shown the efficiency of tumor accumulation of HPMA copolymers is molecular weight dependent.7 In the last decades, active targeting of HPMA copolymer-drug conjugates were achieved by the incorporation of cancer cell-specific ligands, such as carbohydrates, lectins, antibodies, antibody fragments, and peptides. These ligands could bind to antigens or receptors that are either uniquely expressed or overexpressed on the target tumor cells8 and lead to the enhanced uptake of drugs through receptor-mediated endocytosis with concomitant improvement of therapeutic efficacy.9,10 Intravascular cancer cell adhesion plays a significant role on the metastatic process of prostate carcinoma. In addition, increased expression of membrane-bound galectin-3 by tumors has repeatedly been observed to correlate with more poorly differentiated and increased metastatic potential.11 Galectin-3, with high affinity to terminal β-galactopyranose, is involved in multiple biological processes, including adhesion, apoptosis, differentiation, inflammation, and metastasis.12,13 The expression of galectin-3 would be up-regulated in a variety of human cancers, such as prostate carcinoma.14,15 In addition, preferential adhesion of PC3-M human prostate cancer cells to bone marrow endothelial cells was found to be at least in part dependent on galectin-3.16 Therefore, the galectin-3 overexpressed in prostate tumor cells would be a potential binding site for targeting prostate cancer therapy. Phage display is a kind of well-developed laboratory technique used to obtain peptide sequences that can bind to defined proteins, cultured cells, and even inorganic materials.17−19 On the basis of combinatorial phage display, a galectin3-binding peptide, named G3-C12 (the sequence ANTPCGPYTHDCPVKR), has been identified by Suan et al. in 2005. Research on G3-C12 peptide indicated its specific binding to the carbohydrate-recognition domain (CRD) of galectin-3 overexpressed on PC-3 M cells.13 In the previous research, we successfully attached G3-C12 peptide to HPMA copolymers via amide bonds formed by aminolysis of active ester groups on the HPMA copolymers. Importantly, it was documented that G3C12-modified HPMA copolymer-5-fluorouracil conjugates can not only significantly enhanced the drug internalization, subsequent apoptosis-induction and migration of metastatic PC-3 cells but also remarkably improve the in vivo tumor accumulation and anticancer efficacy of therapeutics.20 However, compared with the previous method in which the targeting moieties were amidated onto the HPMA polymer conjugates after copolymerization,20 conjugating polymerizable G3-C12 peptide to the monomers before copolymerization may benefit for the higher conjugated content and affinity of targeting peptide. Moreover, the mechanism of G3-C12 mediation, the effects of the amount of conjugated G3-C12 peptide as well as the molecular weight of HPMA copolymers on their in vitro and in vivo behaviors were still remained unclear. Given the high affinity of G3-C12 peptide for the galectin-3 overexpressed PC-3 cells, this delivery system to the other metastatic prostate carcinoma cell lines, such as DU145 cells, should be investigated to ensure their application in clinic. Therefore, in the present study, G3-C12-modified HPMA copolymers was synthesized by the polymerizable moiety comonomer method and characterized. The effects of the amount of conjugated ligand on the affinity of G3-C12 peptide modified HPMA copolymers to galectin-3 overexpressed prostate cells were investigated. The intracellular uptake and

2. METHODS 2.1. Materials. Fluorescein isothiocyanate (FITC) was purchased from Acros organics. L-Tyrosinamide hydrochloride (Tyr·NH2) was purchased from Bachem Americas, Inc. (Torrance, CA). The galectin-3 binding peptide (G3-C12) was synthesized by CP Biochem Co., Ltd. (Sichuan, China). All other chemicals and reagents were of analytical grade. 2.2. Synthesis and Characterization of G3-C12Modified HPMA Copolymers. 2.2.1. Synthesis of Comonomers. Comonomers {N-(2-hydroxypropyl)methacrylamide (HPMA),21 N-methacryloyl glycyl-glycyl-p-nitrophenyl ester (MA-GG-ONp),22 N-methacryloyl glycyl-glycyl-(G3-C12) (MA-GG-(G3-C12),23 N-methacryloyl tyrosinamide (MATyr·NH2),24 and methacryloyl aminopropyl fluorescein-5isothiocyanate (MAP-FITC)25} were synthesized according to established protocols. Among these comonomers, MA-GGONp and MA-Tyr·NH2 were used for targeting peptide attachment and 131I labeling, respectively. 2.2.2. Synthesis and Characterization of G3-C12-Modified HPMA Copolymers. The G3-C12-modified HPMA copolymers (PG1 and PG2) were synthesized by random radical precipitation copolymerization according to the established procedures.26 Briefly, HPMA, MA-GG-(G3-C12) and other necessary monomers were dissolved in methanol/DMSO mixture with AIBN as the initiator. The solution was purged with nitrogen and stirred at 50 °C for 24 h. After evaporating the solvent, the residue was subsequently dissolved in methanol and precipitated with ether. The loading efficiency of FITC and Tyr·NH2 were determined by UV−vis assay at 492 and 274 nm, respectively. The molecular weight and polydispersity of the conjugates were estimated by size exclusion chromatography on a Superose 200 10/300GL analytical column (Amersham Biosciences, NJ). Meanwhile, the G3-C12 content of the conjugates was determined by amino acid analysis (Commonwealth Biotech. Inc., VA). 2.2.3. 131I-Labeling of HPMA Copolymers. The polymer precursor containing Tyr·NH2 was labeled with 131I in tubes smeared by Idogen. Briefly, copolymers (3 mg) were dissolved into phosphate buffer solution (PBS, 200 μL), and then 3 μL of 131 I solution (62.5 μCi/μL) was added. The mixture was incubated at room temperature for 20 min. Then the 131Ilabeled conjugates were purified by eluting through a Sephadex LH-20 column using saline as eluent. 2.3. In Vitro Biological Characterization. 2.3.1. Cell Culture. Prostate carcinoma cell lines (PC-3 and DU145) were cultured in Dulbecco’s Modified Eagle Media: Nutrient Mixture F-12 (DMEM/F12, 1:1) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 U/mL) at 37 °C in a 5% CO2/95% air atmosphere. Cells were subcultured by disaggregating with Trypsin (0.25%, w/v)EDTA (0.02%, w/v) in buffer phosphate solution (PBS, pH7.4). All experiments were performed on cells in the exponential growth phase. 2.3.2. Expression Level of Galectin-3 in Human Prostate Cancer Cells by qPCR. PC-3 or DU145 cells were seeded in 12well plates at a density of 1 × 105 cells per well and incubated for 24 h. Then the cells were incubated with the nonmodified (pHPMA) and G3-C12-modified (PG1 and PG2) HPMA copolymers at 37 °C for 2 h, respectively. After removing the B



Article

supernatant, the cells were washed with sterile diethyl pyrocarbonate (DEPC) solution and then disrupted by trizol. The lysates were collected, and 200 μL of chloroform was added to separate the total RNA, which was dissolved in the upper aqueous phase, from proteins. After being precipitated by the addition of isopropyl alcohol, the RNA was washed with 75% ethanol solution, preserved in sterile DEPC solution at −80 °C. Then, according to the reverse transcription kit, the cDNA was synthesized and amplified. Finally, the expression level of β-actin and galectin-3 in PC-3 and DU145 cells was quantitated by qPCR. The gene sequence of β-actin and galectin-3 is shown in Table 1.

supernatant were determined as in the above-mentioned method, respectively. 2.4. In Vivo Study. 2.4.1. Animals. Male immunodeficient balb/c mice (aged 4−6 weeks) were purchased from Animal Center of the Institute of West China Medical Center. Animals were housed in accordance with approved guidelines and were provided with food and water. 2.4.2. 131I-SPECT-Imaging and Biodistribution Studies. The biodistribution of 131I-labeled HPMA copolymers (131IpHPMA, 131I-PG1, and 131I-PG2) was estimated by 131ISPECT-imaging and pharmacokinetic tests. The solid tumor was obtained by subcutaneous (s.c.) injection of PC-3 cells (1 × 107) into the shaven right ridge of mice. Within 14 days after the inoculation, the tumors grew up to a volume of 300−400 mm3. Then the animals were fed with sodium iodide solution (0.9%, w/v) for 2 days prior to the following experiments. The animals were randomized into three groups (3 mice/ group), and intravenously (i.v.) administered with 131I-pHPMA, 131 I-PG1, or 131I-PG2 (100−125 μCi per mouse) via tail vein. Sequential 5 min scintigrams were obtained at special times (2 h, 24 h, 48 h, and 72 h) post-intravenous administration (p.i.) with a DSX-LI dual head gamma camera with a high energy allpurpose collimator (SMV, Twinsburg, OH). Time-dependent biodistribution study was carried out at 2 h, 24 h, 48 h and 72 h postadministration. After the animals were euthanized, blood samples were collected from fossa orbitalis, and then samples from the heart, lung, liver, spleen, kidney, and tumor tissues were obtained. These organs and tissues were washed with saline, weighed, and gamma-counted (1470 Automatic Gamma Counter, PerkinElmer Wizard). The percentage-injected dose per gram tissue (% ID/g) was calculated. The study was performed with three mice per group. 2.5. Statistical Analysis. Data were presented as mean ± SD. Comparison among three or more groups were evaluated by one way analysis of variance (ANOVA) followed by t test. A value of p < 0.05 was considered to be significant.

Table 1. Primer Sequence of Gene gene

primer sequence

galectin-3

forward 5′-AGCTTATCCCGGAGCACCTGCACC-3′ reverse 5′-GCAGGGTAGGCTCCGGTGGCAC-3′ forward 5′-ATCATGAAGTGTGACGTGGAC-3′ reverse 5′-AACCGACTGCTGTCACCTTCA-3′

β-actin

2.3.3. Binding of G3-C12-Modified HPMA Copolymers to Galectin-3 Expressed on Human Prostate Cancer Cells. To investigate the affinity of G3-C12 peptide with different tumor cells, competitive binding tests between the specific galectin-3 binding agent, AlexaFluoro-488-labeled asialofetuin (ASF), and free or conjugated G3-C12 were performed on PC-3 or DU145 cells. Nonmodified (pHPMA) and G3-C12-modified (PG1 and PG2) HPMA copolymers were chosen as the samples. After incubation at 37 °C for 2 h, the supernatant was removed and the cells were washed with PBS. Then the AlexaFluoro-488 labeled ASF (10 mM Tris with pH7.4, 100 μL/well) was incubated with cells at 37 °C for 1 h. Finally, the cells were disrupted by 1% Triton X-100, and the total protein was harvested by centrifugation (2000 rpm, 3 min). According to the BicinChoninic Acid (BCA) protein kit, the protein content in the supernatant was determined. Otherwise, the fluorescence intensity was measured by Scientific Fluoroskan Ascent FL (Thermo, U. S. A.). 2.3.4. Uptake of G3-C12-Modified HPMA Copolymers in Human Prostate Cancer Cells. PC-3 or DU145 cells were incubated with FITC-labeled nonmodified (FITC-P) and G3C12-modified (FITC-PG1 and FITC-PG2) HPMA copolymers at 37 °C for 2 h, respectively. After removing the supernatant, the cells were washed with PBS and then disrupted by 1% Triton X-100. The cell lysate was collected into Eppendorf tubes and centrifuged (2000 rpm, 3 min). The fluorescence intensity and protein content in the supernatant were determined as in the above-mentioned method, respectively. 2.3.5. Internalization Mechanism of G3-C12-Modified HPMA Copolymers in Human Prostate Cancer Cells. In order to study the internalization pathway of G3-C12-modified HPMA copolymers on PC-3 and DU145 cells, the cells were incubated with chlorpromazine (10 μg/mL), filipin (100 μg/ mL), or sodium azide solution (100 mM) for 1 h before the addition of FITC-labeled nonmodified (FITC-P) or G3-C12modified (FITC-PG1 and FITC-PG2) HPMA copolymers. After incubation of copolymers at 37 °C for 2 h, the supernatant was removed. The cells were washed with PBS and then disrupted by 1% Triton X-100. The cell lysate was collected into Eppendorf tubes and centrifuged (2000 rpm, 3 min). The fluorescence intensity and protein content in the

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of G3-C12Modified HPMA Copolymers. The synthetic method used for attachment of targeting moieties should be carefully screened because it has an impact on the biorecognition of the HPMA copolymer-drug conjugates. Normally, there were two kinds of methods to achieve the modification on HPMA polymeric backbone. Method A: A polymer precursor containing functional groups was synthesized at first. Then drugs and targeting moieties were conjugated to the macromolecular carriers via suitable reaction, such as ester condensation reaction, which had been widely used. We had documented that the molecular weight (Mw) of HPMA copolymers bearing G3-C12 peptide by Method A was 31.4 kDa with polydispersity index (PDI) of 2.44 and the ligand content was about 2.4 mol %.17 The G3-C12-modifided copolymers had been proved a higher tumor accumulation coupled with a faster clearance from blood circulation on PC-3 tumor-bearing mice model, compared with nonmodified ones. However, increasing the conjugated G3-C12 peptide contents on HPMA polymeric backbone cannot be achieved by this method. Therefore, we focused the study on the other synthetic route. Method B: Monomers were prepared by the conjugation of functional molecules, such as drugs, FITC, or ligands, to the spacers, and then the copolymer conjugates were synthesized at C



Article

Otherwise, the molecular weights (Mw) of all synthetic copolymers were below the renal threshold (40 kDa), which ensured the clearance of macromolecular biomaterials from the body. Regarding to the nonmodified copolymers, lower Mw of FITC-P (16.9 kDa) was observed compared with pHPMA (29.4 kDa). This was resulted from the activated ester ONp in FITC-P. It had been demonstrated by Ghandehari et al. that the absence of MA-GG-ONp during the synthesis of HPMA copolymers resulted in high molecular weight copolymers as ONp acts as a free radical quenching agent.27 Besides, the activity and stability of terminal active ester were also the key points in the process of modification of targeting peptide on copolymers in Method A. Compared with Method B, it required rigorous and anhydrous conditions, which was not easily controlled. Consequently, the application of MA-GG(G3-C12) in Method B might be more beneficial to the G3C12 peptide modification. Interestingly, though the G3-C12 contents in the copolymers elevated with the increasing of feed molar ratio of MA-GG(G3-C12), the Mw of G3-C12-modified copolymers decreased by 9 kDa with higher G3-C12 contents. It had been also proved by David et al. that the high content of G3-C12 monomer can impede the reactivity of the vinyl monomer, resulting in lower molecular weight of copolymers.23 That may be the evidence that the spatial structure of the side-chain had impact on the free radical polymerization reactions. 3.2. Expression of Galectin-3 in Human Prostate Cancer Cells by qPCR. A previous study showed that the G3-C12 peptide has high affinity to the overexpressed galectin3 on prostate cancer cell PC-3 M.16 However, there were no documents about the effects of the G3-C12 contents on the affinity of G3-C12 modified HPMA copolymers to galectin-3 and the mechanisms. Therefore, two metastasis human prostate cancer cell lines (PC-3 and DU145) were selected as cell models to investigate the mechanism of interaction between G3-C12-modified copolymers and galectin-3 by qPCR. On account of the gene expression of β-actin, galectin-3 was proved to be highly expressed in PC-3 and DU145 cells (Figure 2). Besides, the expression level of galectin-3 gene in PC-3 cells was higher than that in DU145 cells. Compared with the control groups, the addition of nonmodified HPMA copolymers (pHPMA) resulted in no significant changes of galectin-3 gene expression level on both cell lines. However, incubation with G3-C12-modified HPMA copolymer (PG1 and PG2) led to significantly decreased expression of galectin-3 (p < 0.01). The depression level was also related to the attached amount of G3-C12 peptide. The lowest level of galectin-3 expression was induced by PG2 with highest G3-C12 content. Notably, compared with DU145 cells, a higher inhibition of galectin-3 expression was found in PC-3 cells with higher expression of galectin-3. It may be supposed that the G3-C12-modified copolymers could suppress the expression of galectin-3 in targeting cells to block the multiple biological processes, such as adhesion,28 differentiation,29 and metastasis.30 Furthermore, the increased attachment of G3-C12 peptide on HPMA copolymers suggested a more efficient biological effect and therapeutic efficacy. 3.3. Binding of G3-C12-Modified HPMA Copolymers to Galectin-3 Overexpressed on Human Prostate Cancer Cells. Macromolecular copolymers modified with moieties can be actively directed to targeting cells by the interaction between ligands and receptors. Enhanced targeting efficiency can be

a certain proportion of the monomers by random radical precipitation copolymerization. It was suitable for synthesis of conjugates containing a variety of functional side chains when the functional molecules and the linkage were stable during the polymerization process. In this study, a polymerizable moiety monomer, named MA-GG-(G3-C12), was prepared via solidphase synthesis, and then FITC-labeled (FITC-PG1 and FITCPG2) and 131I-labeling (PG1 and PG2) G3-C12-modified copolymers were synthesized by random radical precipitation copolymerization based on the specific monomers (Figure 1).

Figure 1. Synthesis of FITC-labeled or 131I-labeled G3-C12-modified HPMA copolymers. Monomers were prepared by the conjugation of G3-C12 peptide to the spacers, and then the copolymers were synthesized at a certain proportion of the monomers by random radical precipitation copolymerization. HPMA, N-(2-hydroxypropyl)methacrylamide; MA-GG-(G3-C12), N-methacryloyl glycyl-glycyl(G3-C12); G3-C12, ANTPCGPYTHDCPVKR.

Notably, as shown in Table 2, the amount of conjugated G3C12 peptide was significantly increased from 3.3 mol % (FITCPG1) to 5.6 mol % (FITC-PG2) with the feed molar ratio of MA-GG-(G3-C12) increasing from 7.5 mol % to 10 mol %. Table 2. Characteristics of Copolymers

copolymers FITC-P FITC-PG1 FITC-PG2 pHPMA PG1 PG2

MAGG -ONp mol %

MAGG-(G3 -C12) mol %

10 7.5 10 7.5 10

Mw (kDa)

PDI

16.9 28.3 19.5 29.4 29.4 21.0

1.24 1.46 1.33 1.40 1.43 1.45

mol % peptide

mol % FITC Tyr mol % 1.7 1.9 1.9

3.3 5.6 3.4 5.4

1.2 1.6 1.6 D



Article

Figure 2. Expression level of galectin-3 on PC-3 cells (A) and DU145 cells (B) with or without the incubation of nonmodified (pHPMA) and G3C12-modified (PG1 and PG2) HPMA copolymers by qPCR (n = 3, *p < 0.05, **p < 0.01).

achieved through multivalent interactions31 between targets and HPMA copolymer−peptide conjugates containing multiple copies of peptides within a single polymer chain.32 Moreover, the adhesion of PC-3 M human prostate cancer cells to bone marrow endothelial cells was found to be at least in part dependent on galectin-3.16 The binding of G3-C12 peptide and G3-C12-modified HPMA copolymers with PC-3 and DU145 cells were further investigated. It was documented that galectin-3 expressed on endothelial cells may interact with carbohydrate ligands such as Thomsen− Friedenreich glycoantigen (TFAg) on carcinoma cells to mediate tumor cell adhesion and metastasis.33 In addition, asialofetuin (ASF) possesses three O-linked desialylated TFAg disaccharide moieties and three branched asparagine-linked oligosaccharides containing a total of nine N-acetyl-lactosamine sequences.12 That means ASF can specific bind to the galectin3 overexpressed on the prostate cells. Before the evaluation of affinity of conjugated G3-C12 peptide with PC-3 and DU145 cells, the competitive inhibition test between free G3-C12 peptide and AlexaFluoro 488-labeled ASF was carried out. As shown in Figure 3, compared with nontreatment cells, the fluorescence intensity of AlexaFluoro 488-labeled ASF in PC-3

cells was significantly decreased with the addition of free G3C12 peptide (0.064 μg/mL) and dropped down to approximately 40% of original intensity at 0.32 μg/mL of free G3-C12 peptide, suggesting the competitive binding of free G3C12 peptide to galectin-3 overexpressed on PC-3 cells. With regard to DU145 cells, the binding profile experienced the same trend. Moreover, the inhibition ratio of galectin-3-binding by G3-C12 peptide on DU145 cells were about 50% and 70% at 0.064 and 0.32 μg/mL of G3-C12 peptide, respectively. Otherwise, as shown in Figure 4, binding of AlexaFluoro 488labeled ASF to galectin-3 dropped down to approximate 20% in PC-3 cells and 25% in DU145 cells after the incubation of G3C12-modified copolymers. It could be explained by the competitive interaction of conjugated G3-C12 peptide with galectin-3 expressed on the two cell lines. PG2, with higher G3C12 peptide contents, still displayed a superior competitive inhibition to the binding of AlexaFluoro 488-labeled ASF to galectin-3 over PG1 in both cell lines. It was suggested that HPMA copolymers with higher G3-C12 attachment can be more efficiently bound to galectin-3, which was essential to block metastasis by inhibiting tumor cell adhesion and invasiveness34 as well as by inducing tumor cell apoptosis,35 antagonizing endothelial cell proliferation and angiogenesis.36 3.4. Quantitative Uptake Analysis of G3-C12-Modified HPMA Copolymers in Human Prostate Cancer Cells. Generally, the enhanced affinity of multiple ligands may improve the uptake of copolymers by target cells via receptor-mediated endocytosis. Furthermore, the internalization of the drug molecules into cells was the prerequisite for its pharmacological effects. The endocytic uptakes of nonmodified (FITC-P) and G3-C12-modified (FITC-PG1 and FITC-PG2) HPMA copolymers were investigated by BCA protein quantitative analysis. Figure 5A showed that the uptake of FITC-P, FITC-PG1, and FITC-PG2 by PC-3 cells were concentration-dependent processes in the presence or absence of free G3-C12 peptide. The fluorescence intensity of FITC-PG1 and FITC-PG2 was much higher than that of FITC-P at the same concentration (p < 0.01). The uptake of G3-C12-modified copolymers at 0.4 mg of polymer/mL was even higher than that of nonmodified ones at 0.8 mg of polymer/mL. It was demonstrated G3-C12 modification resulted in a more efficient internalization in targeting cells. Moreover, the uptake of FITC-PG2 in PC-3

Figure 3. Binding of AlexaFluoro 488-labeled asialofetuin (ASF) to galectin-3 expressed on PC-3 and DU145 cells incubated with free G3C12 peptide (n = 3). E



Article

Figure 4. Binding inhibition ratio of AlexaFluoro 488-labeled asialofetuin (ASF) to galecting-3 on PC-3 (A) and DU145 (B) cells after the incubation with nonmodified (pHPMA) and G3-C12-modified (PG1 and PG2) HPMA copolymers (n = 3, *p < 0.05, **p < 0.01).

cells was about 4-fold higher than that of FITC-PG1 at the concentration of 0.8 mg of polymer/mL, again indicating that the amount of G3-C12 modification played an important role on the internalization of copolymers. Furthermore, a remarkable decrease of the uptake of FITC-PG1 and FITCPG2 was observed in the presence of free G3-C12 at the range from 0.4 to 0.8 mg of polymer/mL (p < 0.01). It was attributed to the competitive inhibition between the free and conjugated G3-C12 peptide. In terms of DU145 cells (Figure 5B), the uptake profile of FITC-P, FITC-PG1, and FITC-PG2 experienced the same trend with that in PC-3 cells. However, the fluorescence intensity of FITC-PG1 and FITC-PG2 was significantly lower than that in PC-3 cells at the same concentration (p < 0.05), possibly due to the lower expression level of galectin-3, which was in agreement with the results of qPCR determination. The noticeable decrease of uptake in PC-3 and DU145 cells by free G3-C12 peptide again indicated the galectin-3-mediated endocytosis. Furthermore, the higher uptake of FITC-PG2 than FITC-PG1 further demonstrated the important role of the amount of conjugated G3-C12 peptide. 3.5. Internalization Mechanism of G3-C12-Modified HPMA Copolymers in Human Prostate Cancer Cells. Manipulation of the subcellular fate of macromolecular therapeutics may result in more effective results because the activity of many drugs is depended on their subcellular location. The uptake mechanism of polymer-bound drugs may be different than that of the free drug.37−39 The internalization pathways of FITC-P, FITC-PG1, and FITC-PG2 were investigated on PC-3 and DU145 cells. Pretreatment of the cells with filipin was reported to block caveolae-mediated uptake process, wheres chlorpromazine is known to perturb clathrin-mediated endocytosis.40 As shown in Figure 6A, the internalization of three copolymers into PC-3 cells all remarkably decreased by the energy depletion of sodium azide. Compared with that of FITC-P, a noticeable decreased uptake of FITC-PG1 and FITC-PG2 was observed with the incubation of filipin and chlorpromazine, suggesting the involvement of both caveolae- and clathrin-mediated endocytosis. In contrast to FITC-PG1, chlorpromazine showed a much higher inhibition ratio than filipin on the internalization of FITC-PG2, implying that the different amount of conjugated

Figure 5. Quantitative uptake analysis of FITC-P, FITC-PG1, and FITC-PG2 by PC-3 cells (A) and DU145 (B) cells in the presence or absence of free G3-C12 peptide using BCA method. Cells were coincubated with three concentrations of conjugates (0.2, 0.4, and 0.8 mg of polymer/mL) and 200 nM of free G3-C12 peptide (n = 3, *p < 0.05, **p < 0.01).

F



Article

distribution of the three copolymers (Figure 7). At 2 h and 24 h p.i., the tumor signal intensity of 131I-PG1 and 131I-PG2 was

Figure 7. Scintigraphic images of mice bearing PC-3 tumor xenografts after intravenous injection of three radiolabeled copolymers (131IpHPMA, 131I-PG1, and 131I-PG2). T pointed to tumor. K pointed to kidney.

obviously higher than that of 131I-pHPMA. Then the tumor accumulation of 131I-pHPMA declined and was barely even detected among 48 h and 72 h. Otherwise, the accumulation of 131 I-PG1 and 131I-PG2 in kidneys were significantly higher than that of 131I-pHPMA, demonstrating the elimination of G3-C12modified HPMA copolymers by kidney. Quantitative determination was performed to further investigate the biodistribution of G3-C12-modified HPMA copolymers. Tumor tissues and major organs including heart, lung, spleen, liver, and kidney were harvested at 2 h, 24 h, 48 h, and 72 h p.i. (Figure 8 and Table 3). After administration for 2 h, 131I-PG1 (1.60 ± 0.08% ID/g) and 131I-PG2 (1.54 ± 0.06% ID/g) displayed equal tumor accumulation, which was higher than unmodified 131I-pHPMA (1.19 ± 0.04% ID/g, p < 0.05). It was indicated that the fast and enhanced tumor accumulation of copolymers within 2 h may be attributed to the G3-C12 modification. Interestingly, as the time was prolonged from 24 h to 72 h, although 131I-PG2 with higher degree of ligand modification was supposed to have higher affinity toward tumor cells with concomitant increase in tumor site, 131I-PG1 with higher molecular weight displayed better tumor accumulation than 131I-PG2. As the incorporation of higher content of G3C12 peptide could compromise the molecular weight (Mw) of 131 I-PG2 (21.0 kDa) compared with 131I-PG1 (29.4 kDa), it was suggested that more pronounced EPR effect obtained by higher molecular weight of 131I-PG1 could be responsible for this observation. The higher molecular weight of polymer carriers could prevent fast elimination of the drug from the

Figure 6. Quantitative analysis of uptake of FITC-P, FITC-PG1, and FITC-PG2 (0.4 mg/mL of polymer) by PC-3 (A) and DU145 (B) cells with the addition of inhibitors using BCA method (n = 3, *p < 0.05, **p < 0.01).

G3-C12 peptide may lead to different uptake pathways for the internalization of HPMA copolymers. In terms of DU145 cells, the uptake processes of FITC-P, FITC-PG1, and FITC-PG2 were also in an energy-dependent manner (Figure 6B). In addition, the decreased uptake of FITC-PG1 and FITC-PG2 was resulted from the incubation of chlorpromazine, demonstrating the involvement of clathrinmediated endocytosis. Compared with PC-3 cells, there was no remarkable decreased effect by filipin. Followed by the G3-C12mediated membrane-associated endocytosis, the combination of caveolae- and clathrin-mediated endocytosis might be able to account for the more efficient uptake process in PC-3 cells than that in DU145 cells. 3.6. In Vivo Biodistribution Analysis of G3-C12Modified HPMA Copolymers. To evaluate the in vivo behavior of nonmodified and G3-C12-modified copolymers, iodine-131 was labeled onto the polymeric backbone via the tyrosinamide groups. Subsequently, the 131I-labeled copolymers, named 131I-pHPMA, 131I-PG1, and 131I-PG2, were intravenously administrated to mice bearing PC-3 tumor xenografts. At 2 h, 24 h, 48 h, and 72 h post-intravenous injection (p.i.), 131 I-SPECT-scintigrams were obtained to visualize the bioG



Article

Figure 8. Quantification of the tumor and organ concentration of 131I-pHPMA, 131I-PG1, and 131I-PG2 at 2 h (A), 24 h (B), 48 h (C), and 72 h (D) p.i. (n = 3).

Table 3. Tumor Accumulation of copolymer 131

I-pHPMA 131 I-PG1 131 I-PG2 a

131

I-Labeled Copolymersa

2h

24 h

48 h

72 h

1.19 ± 0.04 1.60 ± 0.08b 1.54 ± 0.06b

0.36 ± 0.04 1.04 ± 0.19c 0.64 ± 0.04b

0.30 ± 0.05 0.52 ± 0.09b 0.25 ± 0.03

0.24 ± 0.03 0.34 ± 0.09 0.20 ± 0.01d

n = 3. bp < 0.05 and. cp < 0.01 vs pHPMA. dp < 0.05 vs PG1.

protein-inhibitor geldanamycin, and antiangiogenic agent TNP470, and exhibited desirable in vivo biodistribution, elimination, and metabolism properties. 20 In addition, five HPMA copolymer-based polymeric drug delivery systems have progressed into clinical trials.41,42 In our previous research, 5fluorouracil with 4.73 wt % of drug loading has been attached onto G3-C12 modified HPMA polymeric backbone via enzymatically degradable oligopeptide glycylphenylalanylleucylglycine (GFLF) sequence.20 In other research, doxorubicin with over 10 wt % of drug loading has been linked to the copolymers via pH-susceptible hydrazone linkages.43,44 These spacers proved to be stable in the circulation. For instance, the release ratio of 5-Fu from G3-C12-modified HPMA copolymer-5fluorouracil conjugates (P-(G3-C12)-Fu) in plasma was less than 20% within 12 h, indicating the stability of conjugated 5Fu in HPMA copolymers. However, as the endo/lysosome in cancer cell is rich in degradable enzymes and has an acidic environment with a pH of about 5−6, drug release could be triggered in response to these specific conditions. For example, doxorubicin conjugated to HPMA copolymers via hydrazone linkage showed more than 80% drug release within 48 h at pH 5 mimicking endo/lysosome, whereas less than 20% drug

organism, thus enabling prolonged blood circulation and increased tumor accumulation. The higher amount of tumor and blood concentration of 131I-PG1 over 131I-PG2 in Figure 8 could also support this hypothesis. However, with the same molecular weight of approximately 29 kDa, the higher tumor accumulation of 131I-PG1 than 131IpHPMA was observed from 2 h to 72 h, implying the tumor targeting effects by G3-C12 peptide. Therefore, the targeting ability of G3-C12 peptide could only be exhibited under the circumstance that the Mw was not sacrificed after ligand modification. Apparently, in the case of G3-C12 modified HPMA copolymers, besides the amount of conjugated G3-C12 peptide, the Mw also played an important role on their in vivo tumor localization. Our data suggested that the passively targeted EPR effect, which was significantly affected by the molecular weight, would first drive G3-C12 modified HPMA copolymers from the in vivo circulation to accumulate in the tumor rather than the active ligand targeting. Then the G3-C12 ligand−receptor interaction occurred after the tumor arrival of the copolymers. Nowadays, various types of drug have been conjugated to HPMA copolymers, such as doxorubicin, paclitaxel, heat shock H



Article

Notes

release took place at pH 7.4, mimicking physiological conditions.43 Moreover, it has been demonstrated that the efficacy of HPMA copolymer-based nanomedicines could be enhanced by the incorporation of targeting moieties (peptides) via selectively binding drugs to antigens or receptors unique expressed or overexpressed on the target cells.8 In this the present study, HPMA served as a linear scaffold to which different amounts of G3-C12 peptide were attached as a targeting moiety to Gal-3-expressed cells. Therefore, based on this targeting delivery system, the above-mentioned drug loading methods with specific drug release could also be applied in further study. In terms of therapeutic effect, our previous study showed G3C12-modified HPMA copolymer-5-fluorouracil conjugates (P(G3-C12)-Fu) can not only significantly enhance the drug internalization, apoptosis-induction, and migration of metastatic PC-3 cells but also remarkably improve the in vivo tumor accumulation and anticancer efficacy of therapeutics.20 A superior inhibition of tumor growth (71%, p < 0.01) was observed on P-(G3-C12)-Fu compared with those of 5-Fu (23%, p < 0.05) and HPMA copolymer-5-fluorouracil conjugates (37%, p < 0.05). However, in the present study, we discovered that G3-C12 modification was not the only one important factor that affected the in vivo tumor accumulation of HPMA-based copolymers. Notably, the molecular weight could also have great impact. Though the G3-C12-modified copolymers (131I-PG1 and 131I-PG2) accumulated faster in the tumor than unmodified ones (131I-pHPMA, p < 0.05) within 2 h, 131I-pHPMA with larger Mw showed higher tumor restraint than 131I-PG2 with smaller Mw after 48 h of administration. Therefore, when it comes to future application potential, the effect of targeting ligand should not be overrated, but a balance between ligand modification degree and Mw should also be taken into consideration. Hence, we hypothesized that drug-loaded HPMA copolymer with proper G3-C12 modification, a specific drug release profile, and higher Mw could have great potential feasibility to improve its therapeutic effect.

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS The research described above was supported by the National Natural Science Foundation (81072600).

4. CONCLUSION HPMA copolymers containing various amounts of G3-C12 peptide were successfully synthesized. The higher amount of conjugated G3-C12 peptide displayed a more significant effect on the in vitro affinity of copolymers to galectin-3 overexpressed on PC-3 and D145 cells. Compared with DU145 cells, the higher internalization of G3-C12-modified copolymers was observed in PC-3 cells, which was involved in caveolin- and clathrin-mediated endocytosis as well as the galectin-3mediated uptake. Importantly, the superior tumor accumulation of G3-C12-modified copolymers was further proved on a PC-3 tumor-bearing mice model. It was demonstrated that the amount of conjugated G3-C12 peptide performed a remarkable effect on the in vitro affinity and internalization of HPMA copolymers to the galectin-3 overexpressed prostate carcinoma cells. However, molecular weight and ligand modification all play important roles on their in vivo tumor accumulation.

■

REFERENCES

(1) Bogenrieder, T.; Herlyn, M. Axis of evil: molecular mechanisms of cancer metastasis. Oncogene 2003, 22, 6254−6236. (2) Laufer, M.; Denmeade, S. R.; Sinibaldi, V. J.; Carducci, M. A.; Eisenberger, M. A. Complete androgen blockade for prostate cancer: what went wrong? J. Urol. 2000, 164, 3−9. (3) Gilligan, T.; Kantoff, P. W. Chemotherapy for prostate cancer. Urology 2002, 60, 94−100. (4) Omelyanenko, V.; Kopecková, P.; Gentry, C.; Shiah, J. G.; Kopecek, J. HPMA copolymer -anticancer drug-OV-TL16 antibody conjugates. 1. Influence of the method of synthesis on the binding affinity to OVCAR-3 ovarian carcinoma cells in vitro. J. Drug Target. 1996, 3, 357−373. (5) Liu, J.; Kopecková, P.; Bühler, P.; Wolf, P.; Pan, H.; Bauer, H.; Elsässer-Beile, U.; Kopecek, J. Biorecognition and subcellular trafficking of HPMA copolymer-anti-PMSA antibody conjugates by prostate cancer cells. Mol. Pharm. 2009, 6, 959−970. (6) Kopecek, J.; Kopecková, P. HPMA copolymers: Origins, early developments, present, and future. Adv. Drug Delivery Rev. 2010, 62, 122−149. (7) Seymour, L. W.; Miyamoto, Y.; Maeda, H.; Brereton, M.; Strohalm, J.; Ulbrich, K.; Duncan, R. Influence of molecular-weight on passive tumor accumulation of a soluble macromolecular drug carrier. Eur. J. Cancer. 1995, 31A, 766−770. (8) Allen, T. M. Ligand-targeted therapeutics in anticancer therapy. Nat. Rev. Cancer 2002, 2, 750−763. (9) Jensen, K. D.; Nori, A.; Tijerina, M.; Kopecková, P.; Kopecek, J. Cytoplasmic delivery and nuclear targeting of synthetic macromolecules. J. Controlled Release 2003, 87, 89−105. (10) Lu, Z. R.; Shiah, J. G.; Sakuma, S.; Kopecková, P.; Kopecek, J. Design of novel bioconjugates for targeted drug delivery. J. Controlled Release 2002, 78, 165−173. (11) Bresalier, R. S.; Mazurek, N.; Sternberg, L. R.; Byrd, J. C.; Yunker, C. K.; Nangia-Makker, P.; Raz, A. Metastasis of human colon cancer is altered by modifying expression of the beta-galactoside -binding protein galectin-3. Gastroenterology 1998, 115, 287−296. (12) Zou, J.; Glinsky, V. V.; Landon, L. A.; Matthews, L.; Deutscher, S. L. Peptides specific to the galectin-3 carbohydrate recognition domain inhibit metastasis-associated cancer cell adhesion. Carcinogenesis 2005, 26, 309−318. (13) Inohara, H.; Akahani, S.; Koths, S.; Raz, A. Interactions between galectin-3 and Mac-2-binding protein mediate cell-cell adhesion. Cancer Res. 1996, 56, 4530−4534. (14) Wang, L.; Inohara, H.; Pienta, K. J.; Raz, A. Galectin-3 is a nuclear matrix protein which binds RNA. Biochem. Biophys. Res. Commun. 1995, 217, 292−303. (15) Meromsky, L.; Lotan, R.; Raz, A. Implications of endogenous tumor cell surface lectins as mediators of cellular interactions and lung colonization. Cancer Res. 1986, 46, 5270−5275. (16) Deutscher, S. L.; Figueroa, S. D.; Kumar, S. R. Tumor targeting and SPECT imaging properties of an (111)In-labeled galectin-3 binding peptide in prostate carcinoma. Nucl. Med. Biol. 2009, 36, 137− 146. (17) Smith, G. P.; Petreno, V. A. Phage display. Chem. Rev. 1997, 97, 391−410. (18) Rodi, D. J.; Janes, R. W.; Sanganee, H. J.; Holton, R. A.; Wallace, B. A.; Makowski, L. Screening of a library of phage-displayed peptides identifies human bcl-2 as a taxol-binding protein. J. Mol. Biol. 1999, 285, 197−203. (19) Meyers, S. R.; Khoo, X.; Huang, X.; Walsh, E. B.; Grinstaff, M. W.; Kenan, D. J. The development of peptide-based interfacial

AUTHOR INFORMATION

Corresponding Author

*Y. Huang. E-mail: [email protected]. Tel.: +86-28-85501617. Fax: +86-28-8550-1617. Address: West China School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, People’s Republic of China. I



Article

biomaterials for generating biological functionality on the surface of bioinert materials. Biomaterials 2009, 30, 277−286. (20) Yang, Y.; Zhou, Z.; He, S.; Fan, T.; Jin, Y.; Zhu, X.; Chen, C.; Zhang, Z. R.; Huang, Y. Treatment of prostate carcinoma with (galectin-3)-targeted HPMA copolymer-(G3-C12)-5-Fluorouracil conjugates. Biomaterials 2012, 33, 2260−2271. (21) Ulbrich, K.; Subr, V.; Strohalm, J.; Plocová, D.; Jelínková, M.; Ríhová, B. Polymeric drugs based on conjugates of synthetic and natural macromolecules I. Synthesis and physico-chemical characterization. J. Controlled Release 2000, 64, 63−79. (22) Yuan, F.; Chen, F.; Xiang, Q. Y.; Qin, X.; Zhang, Z. R.; Huang, Y. Synthesis and characterization of HPMA copolymer-5-FU conjugates. Chin. Chem. Lett. 2008, 19, 137−140. (23) Kopansky, E.; Shamay, Y.; David, A. Peptide-directed HPMA copolymer-doxorubicin conjugates as targeted therapeutics for colorectal cancer. J. Drug Target. 2011, 19, 933−943. (24) Ruttekolk, I. R.; Duchardt, F.; Fischer, R.; Wiesmüller, K. H.; Rademann, J.; Brock, R. HPMA as a scaffold for the modular assembly of functional peptide polymers by native chemical ligation. Bioconjug Chem. 2008, 19, 2081−2087. (25) Rademaker-Lakhai, J. M.; Terret, C.; Howell, S. B.; Baud, C. M.; De Boer, R. F.; Pluim, D.; Beijnen, J. H.; Schellens, J. H.; Droz, J. P. A phase I and pharmacological study of the platinum polymer AP5280 given as an intravenous infusion once every 3 weeks in patients with solid tumors. Clin. Cancer Res. 2004, 10, 3386−3395. (26) Nan, A.; Ghandehari, H.; Hebert, C.; Siavash, H.; Nikitakis, N.; Reynolds, M.; Sauk, J. J. Water-soluble polymers for targeted drug delivery to human squamous carcinoma of head and neck. J. Drug Target. 2005, 13, 189−197. (27) Greish, K.; Ray, A.; Bauer, H.; Larson, N.; Malugin, A.; Pike, D.; Haider, M.; Ghandehari, H. Anticancer and antiangiogenic activity of HPMA copolymer-aminohexylgeldanamycin-RGDfK conjugates for prostate cancer therapy. J. Controlled Release 2011, 151, 263−270. (28) Inohara, H. A. Functional evidence that cell surface galectin-3 mediates homotypic cell adhesion. Cancer Res. 1995, 55, 3267−3271. (29) Akahani, S.; Nangia-Makker, P.; Inohara, H.; Kim, H. R.; Raz, A. Galectin-3: a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family. Cancer Res. 1997, 57, 5272−5276. (30) Matarrese, P.; Fusco, O.; Tinari, N.; Natoli, C.; Liu, F. T.; Semeraro, M. L.; Malorni, W.; Iacobelli, S. Galectin-3 overexpression protects from apoptosis by improving cell adhesion properties. Int. J. Cancer. 2000, 85, 545−554. (31) Marradi, M.; Martín-Lomas, M.; Penadés, S. Glyconanoparticles polyvalent tools to study carbohydrate-based interactions. Adv. Carbohydr.: Chem. Biochem. 2010, 64, 211−290. (32) Tang, A.; Kopeiková, P.; Kopeckevá, J. Binding and cytotoxicity of HPMA copolymer conjugates to lymphocytes mediated by receptor-binding epitopes. Pharm. Res. 2003, 20, 360−367. (33) Glinsky, V. V.; Glinsky, G. V.; Rittenhouse-Olson, K.; Huflejt, M. E.; Glinskii, O. V.; Deutscher, S. L.; Quinn, T. P. The role of Thomsen−Friedenreich antigen in adhesion of human breast and prostate cancer cells to the endothelium. Cancer Res. 2001, 61, 4851− 4857. (34) Le Marer, N.; Hughes, R. C. Effects of the carbohydrate-binding protein galectin-3 on the invasiveness of human breast carcinoma cells. J. Cell Physiol. 1996, 168, 51−58. (35) Yu, F.; Finley, R. L.; Raz, A.; Kim, H. R. Galectin-3 translocates to the perinuclear membranes and inhibits cytochrome c release from the mitochondria. A role for synexin in galectin-3 translocation. J. Biol. Chem. 2002, 277, 15819−15827. (36) Nangia-Makker, P.; Honjo, Y.; Sarvis, R.; Akahani, S.; Hogan, V.; Pienta, K. J.; Raz, A. Galectin-3 induces endothelial cell morphogenesis and angiogenesis. Am. J. Pathol. 2000, 156, 899−909. (37) Nori, A.; Kopecek, J. Intracellular targeting of polymer-bound drugs for cancer chemotherapy. Adv. Drug Delivery Rev. 2005, 57, 609−636. (38) Tijerina, M.; Kopecková, P.; Kopecek, J. Correlation of subcellular compartmentalization of HPMA copolymer-Mce6 con-

jugates with chemotherapeutic activity in human ovarian carcinoma cells. Pharm. Res. 2003, 20, 728−737. (39) Tijerina, M.; Kopecková, P.; Kopecek, J. Mechanism of cytotoxicity in human ovarian carcinoma cells exposed to free Mce6 or HPMA copolymer-Mce6 conjugates. Photochem. Photobiol. 2003, 77, 645−652. (40) Rejman, J.; Conese, M.; Hoekstra, D. Gene transfer by means of lipo- and polyplexes: role of clathrin and caveolae-mediated endocytosis. J. Liposome Res. 2006, 16, 237−247. (41) Duncan, R. Polymer conjugates as anticancer nanomedicines. Nat. Rev. Cancer. 2006, 6, 688−701. (42) Ríhová, B.; Kubácková, K. Clinical implications of N-(2hydroxypropyl)methacrylamide copolymers. Curr. Pharm. Biotechnol. 2003, 4, 311−322. (43) Li, L.; Yang, Q. Q.; Zhou, Z.; Zhong, J. J.; Huang, Y. Doxorubicin-loaded, charge-reversible, folate-modified HPMA copolymer for active cancer cell targeting. Biomaterials 2014, 35, 5171−5187. (44) Zhou, Z.; Li, L.; Yang, Y.; Xu, X. L.; Huang, Y. Enhanced tumor targeting by cross-linked HPMA copolymer micelles with pHsensitivity and biodegrability. Biomaterials 2014, 35, 6622−6635.

J


Targeting Prostate Carcinoma by G3-C12 Peptide Conjugated

Recommend Documents