Targeted delivery of cisplatin by LHRH-peptide conjugated dextran nanoparticles suppresses breast cancer growth and metastasis

Acta Biomaterialia 18 (2015) 132–143

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Targeted delivery of cisplatin by LHRH-peptide conjugated dextran nanoparticles suppresses breast cancer growth and metastasis Mingqiang Li a,c, Zhaohui Tang a, Yu Zhang a,c, Shixian Lv a,c, Quanshun Li b, Xuesi Chen a,⇑ a

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130012, PR China c University of Chinese Academy of Sciences, Beijing 100049, PR China b

a r t i c l e

i n f o

Article history: Received 16 August 2014 Received in revised form 12 December 2014 Accepted 23 February 2015 Available online 28 February 2015 Keywords: Breast cancer Metastasis Targeted drug delivery Chemotherapy Cisplatin

a b s t r a c t The metastasis of breast cancer is the leading cause of cancer death in women. In this work, an attempt to simultaneously inhibit the primary tumor growth and organ-specific metastasis by the cisplatin-loaded LHRH-modified dextran nanoparticles (Dex-SA-CDDP-LHRH) was performed in the 4T1 orthotopic mammary tumor metastasis model. With the rationally designed conjugation site of the LHRH ligand, the Dex-SA-CDDP-LHRH nanoparticles maintained the targeting function of LHRH and specifically bound to the LHRH-receptors overexpressed on the surface of 4T1 breast cancer cells. Therefore, the Dex-SACDDP-LHRH nanoparticles exhibited improved cellular uptake and promoted cytotoxicity, when compared with the non-targeted Dex-SA-CDDP nanoparticles. Moreover, both the non-targeted and targeted nanoparticles significantly decreased the systemic toxicity of CDDP and increased the maximum tolerated dose of CDDP from 4 to 30 mg kg1. Importantly, Dex-SA-CDDP-LHRH markedly enhanced the accumulation of CDDP in the injected primary tumor and metastasis-containing organs, and meanwhile significantly reduced the nephrotoxicity of CDDP. Dose-dependent therapeutic effects further demonstrated that the CDDP-loaded LHRH-decorated polysaccharide nanoparticles significantly enhanced the antitumor and antimetastasis efficacy, as compared to the non-targeted nanoparticles. These results suggest that Dex-SA-CDDP-LHRH nanoparticles show great potential for targeted chemotherapy of metastatic breast cancer. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Breast cancer is the most common malignancy and the second leading cause of cancer-related death in women [1]. Surgical resection in combination with adjuvant therapy is efficient at the early stages of disease, but subsequent relapse and metastatic spread to vital organs such as lung, liver, and bone often occur and lead to cancer death in women with more than a million newly diagnosed cases annually worldwide [2,3]. Tumor metastasis is a multistep process, which can be defined as the ability of malignant tumor cells to escape from the primary tumor, migrate, invade surrounding tissues, enter the vasculature, circulate, reach secondary sites and establish metastatic foci [4–6]. More than 60% of the malignant tumors have been in progression of metastasis when they are first diagnosed, while other tumor patients are also subject to tumor metastasis during treatment and even after first recovery for several years [5]. Therefore, tumor metastasis is responsible ⇑ Corresponding author. Tel./fax: +86 431 85262112. E-mail address: [email protected] (X. Chen). http://dx.doi.org/10.1016/j.actbio.2015.02.022 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

for as much as 90% of cancer-associated deaths and is almost inaccessible by surgical operation or radiotherapy [7,8]. Although the inhibition of tumor metastasis is of great significance in cancer therapy and effective progress in the treatment of metastatic breast cancer is expected to have great benefits for women, the development of antimetastasis strategy is still in its infancy stage and the efficient antimetastasis drugs are still much in demand. Targeting a metastatic lesion within a large population of normal cells remains a significant challenge. In addition to reducing the toxicity and altering biodistribution via the enhanced permeability and retention (EPR) effect, further increasing the tumor accumulation of anticancer drugs via active targeting ligands also plays an important role in cancer therapy [9,10]. Luteinizing hormone-releasing hormone (LHRH, also known as gonadotropinreleasing hormone, GnRH) is a hormonal decapeptide produced by the hypothalamus, which regulates the pituitary–gonadal axis and thus reproduction [11]. Overexpression of the LHRH receptors has been demonstrated in many tumors including breast (about 50%), ovarian and endometrial (about 80%), and prostate (about 90%) tumors, while their expression is scarce in healthy tissues,

M. Li et al. / Acta Biomaterialia 18 (2015) 132–143

which makes them an ideal tumor target for constructing nanoparticles for targeted therapy of gonadal tumors [12,13]. Recently, the Bronich group has reported the first example of LHRH-targeted CDDP delivery for ovarian cancer treatment [12]. Leuschner et al. have developed LHRH-conjugated magnetic iron oxide nanoparticles for detection of breast cancer metastases, based on the interaction of LHRH-conjugated nanoparticles with the LHRHpositive MDA-MB-435S cells [14]. However, the application of nanodrug delivery system with LHRH peptide as the targeting ligand for the chemotherapy of metastatic breast cancer has not been extensively studied yet. We have recently developed the LHRH-peptide conjugated dextran nanoparticles for targeted delivery and controlled release of cisplatin (CDDP) [15]. Dextran (Dex) was selected as a structural molecule based on its excellent aqueous solubility, wide availability, the known biocompatibility in medicine and food, ease of modification, and FDA approval in parenteral formulation [16,17]. The targeted nanoparticles (Scheme 1A, Dex-SA-CDDP-LHRH)

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exhibit a negative surface charge (17 mV), condense into welldefined nanoparticles with uniform size (Rh: 22 nm), and release drug at a controlled rate [15]. Both the non-targeted (Scheme 1A, Dex-SA-CDDP) and targeted nanoparticles can significantly prolong the blood circulation of CDDP and reduce the systemic toxicity attributed to the delayed and sustained drug release behavior [15]. More importantly, the LHRH-targeted nanoparticles lead to significantly higher drug internalization in MCF-7 tumor cells in vitro and enhanced accumulation in MCF-7 xenograft tumors in vivo, compared with the non-targeted counterparts [15]. Furthermore, systemic delivery of the targeted nanoparticles carrying CDDP via intravenous injection can significantly retard tumor growth in MCF-7 tumor-bearing mice compared to the non-targeted nanoparticles and free CDDP [15]. The purpose of this study was to expand the functionality of the CDDP-loaded polysaccharide nanoparticles decorated with LHRH ligand, and provide references for the next preclinical and clinical studies. Herein, we report the therapeutic effects in the orthotopic

Scheme 1. (A) Schematic illustration of the chemical composition and physicochemical properties of Dex-SA-CDDP and Dex-SA-CDDP-LHRH nanoparticles. (B) Schematic illustration of preparing orthotopic mammary tumor metastasis model and the corresponding therapeutic effects of different administrations.

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mammary tumor metastasis model after the treatment with free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH, and compare their antimetastatic activities. To the best of our knowledge, this was the first example to apply LHRH-targeted polysaccharide nanoparticles as the carrier for the targeted cisplatin chemotherapy of metastatic breast cancer. Comparisons of maximum tolerated dose, in vitro cellular uptake, and in vivo biodistributions in the normal Balb/c mice and 4T1 tumor-bearing mice were also carried out to confirm the safety and specificity of the targeted polysaccharide nanoparticles.

were seeded in 96-well plates (8  103 cells per well) in 100 lL of culture medium and incubated at 37 °C in a 5% CO2 atmosphere for 24 h. The culture medium was replaced with 200 lL of fresh medium containing Dex-SA, free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH. The cells were subjected to MTT assay after being incubated for another 24 or 48 h. The absorbency of the solution was measured on a Bio-Rad 680 microplate reader at 490 nm. The relative cell viability was determined by comparing the absorbance at 490 nm with control wells containing only cell culture medium. Data were presented as means ± standard deviation (n = 6).

2. Experimental section 2.6. Evaluation of maximum tolerated dose 2.1. Materials

Dex-SA-CDDP and Dex-SA-CDDP-LHRH nanoparticles were synthesized according to the previous report [15]. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma), and nitric acid (68 vol.%, Beijing Chemical Factory, China) were used without further purification. Purified deionized water was prepared by the Milli-Q plus system (Millipore Co., Billerica, MA, USA). 2.2. Animals Male Kunming mice (at 5–6 weeks of age) were provided by Laboratory Animal Center of Jilin University. Female Balb/c mice at 5–6 weeks of age were obtained from Beijing HFK Bioscience Co., Ltd. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and all procedures were approved by the Animal Care and Use Committee of Jilin University. 2.3. Cell lines and cell culture 4T1 cells (murine breast cancer) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI medium 1640 (Gibco) supplemented with 10% FBS (heat-inactivated, Hyclone), penicillin (50 U mL1), streptomycin (50 U mL1), glucose (2.5 g L1) and sodium pyruvate (0.11 g L1) at 37 °C in a humid atmosphere with 5% CO2. The cell density was determined before each experiment using a hemocytometer.

Male Kunming mice were used to evaluate the maximum tolerated dose of free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH. All groups (n = 3) received a single dose by intravenous injection. Six groups of mice received free CDDP (1, 4, 10, 15, 20 and 25 mg kg1), or Dex-SA-CDDP and Dex-SA-CDDP-LHRH (4, 10, 20, 30, 40 and 50 mg kg1 CDDP), respectively. The body weights and physical states of all the mice were monitored for a period of 10 d. The maximum tolerated dose was defined as the allowance of a median body weight loss of 20% and caused neither death due to the toxic effects nor remarkable changes in the general signs within 10 d after administration [18]. 2.7. Drug distribution in the tumor tissues The biodistribution study was carried out in both normal Balb/c mice and 4T1 tumor-bearing mice. For the biodistribution in tumor-bearing mice, a mouse mammary carcinoma model was generated by the subcutaneous injection of 4T1 cells (1.5  106) into the mammary fat pad of the mice (female Balb/c mice, 5–6 weeks old). When the tumor volumes reached approximately 400 mm3, the mice were administered intravenously with free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH nanoparticles at an equivalent CDDP dose of 5 mg kg1 (n = 3 per group). The mice were sacrificed after 2, 10 and 24 h. Then the tumor tissues and major organs (heart, liver, spleen, lung and kidney) were excised, washed 3 times with cold saline, dried on filter paper, weighed and cut into small pieces. The amount of platinum in the tissues was determined by ICP-MS as previously reported [19]. The biodistribution study in normal mice was carried out in female Balb/c mice according to similar procedures described above.

2.4. Cellular uptake 2.8. In vivo inhibition of tumor growth and metastasis 4T1 cells were seeded in 6-well plates with a density of 3  105 cells per well in 2 mL of culture medium and incubated for 24 h, and then the original medium was replaced with free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH (at a final CDDP concentration of 12.0 lM) containing culture medium. For the LHRH receptor blocking study, 4T1 cancer cells were first incubated with free LHRH (20 lM) for 1 h, followed by co-incubation with Dex-SACDDP-LHRH. After incubation for 5 h at 37 °C, the cells were washed 5 times with cold phosphate buffered saline (PBS). The cells were trypsinized and cell numbers were counted. Then, the cells were digested with nitric acid (68 vol.%) at 70 °C for 12 h. The platinum concentration was measured by inductively coupled plasma mass spectrometer (ICP-MS, Xseries II, Thermoscientific, USA). The reported results of the sample were the average of three replicates. 2.5. Cytotoxicity assay The cytotoxicities of Dex-SA, free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH were evaluated by MTT assay. The 4T1 cells

The metastatic mammary carcinoma model was generated by the subcutaneous injection of 4T1 cells (1.5  106) into the mammary fat pad of the mice. When the tumor volumes were approximately 50 mm3, the mice were randomly divided into seven groups and then treated with PBS, free CDDP (2.0 mg kg1), free CDDP (5.0 mg kg1), Dex-SA-CDDP (5.0 mg kg1 on CDDP basis), Dex-SA-CDDP (10.0 mg kg1 on CDDP basis), Dex-SA-CDDP-LHRH (5.0 mg kg1 on CDDP basis) and Dex-SA-CDDP-LHRH (10.0 mg kg1 on CDDP basis), respectively, by intravenous injection on days 0, 4, and 8. The treatment efficacy and systemic toxicity were assessed by measuring the tumor volume and body weight, respectively. The tumor volume and tumor suppression rate were calculated by the following formula: 2

Tumor volume ðVÞ ¼ a  b =2 Tumor inhibition rate ðTIR; %Þ ¼ ½ðW c  W x Þ=W c Þ  100% a and b were the longest and shortest diameter of the tumors measured by vernier caliper. Wc represented the average weight of

M. Li et al. / Acta Biomaterialia 18 (2015) 132–143

tumors in the control group, while Wx represented the average weight of tumors in the treatment group. To further investigate the antimetastasis efficacy of Dex-SACDDP-LHRH on metastatic 4T1 tumor in late stage, four groups of mice (n = 6) were subcutaneously injected of 4T1 cells (3  106) into the mammary fat pad. As 4T1 tumors grew to a volume of 400– 500 mm3 (15 days after inoculation), the mice were administered intravenously with PBS, free CDDP (3.0 mg kg1), Dex-SA-CDDP (10.0 mg kg1 on CDDP basis), and Dex-SA-CDDP-LHRH (10.0 mg kg1 on CDDP basis) on days 15, 19, and 23. The antimetastasis efficacy was assessed by measuring the weight of the sentinel lymph node and the conventional histological analysis. 2.9. Histological and immunohistochemical analyses The mice were sacrificed at day 20, and the tumors and major organs (liver, lung, kidney and lymph node) were collected immediately, fixed in 4% PBS buffered paraformaldehyde overnight, and then embedded in paraffin. The paraffin-embedded tumors and organs were cut at 5 lm thickness, and stained with hematoxylin and eosin (H&E) to assess histological alterations by microscope (Olympus CX31). Immunohistochemistry was performed as described previously [20,21]. Rabbit monoclonal primary antibody for cleaved PARP1 (Abcam, Cambridge, MA, USA) and PV-6000 two-step immunohistochemistry kit (polymer detection system for immuno-histological staining; Zhongshan Goldbridge Biotechnology, Beijing, China) were used in this study.

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consistent with our previous study in MCF-7 cells and similar to what has been reported in A2780 cells by Kabanov et al. [12,15]. The biocompatibility of Dex-SA was examined prior to platinum chelation regarding the viability of 4T1 cells. The carboxylic ligand functionalized polysaccharide carrier was nontoxic up to the highest testing concentration of 1 g L1, indicating its excellent biocompatibility (Fig. 1B). After being incubated with free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH, the 4T1 cell viability was obviously inhibited in a dose- and time-dependent manner (Fig. 1C and D). The viability of cells treated with free CDDP decreased sharply, with the 50% growth inhibitory concentration (IC50) of 8.95 and 5.43 lmol Pt L1 after 24 and 48 h incubation, respectively. Small molecule drugs are typically internalized via passive diffusion through the cell membrane [24]. However, the uptake of polymeric micelles is mediated by endocytosis and the encapsulated drug must undergo a slow process of release after the cellular internalization [25]. Therefore, free CDDP produced higher cytotoxicity than Dex-SA-CDDP and Dex-SA-CDDP-LHRH at the same concentration. In addition, compared with Dex-SA-CDDP (24 h: IC50 = 87.95 lmol Pt L1; 48 h: IC50 = 34.68 lmol Pt L1), Dex-SA-CDDP-LHRH (24 h: IC50 = 66.27 lmol Pt L1; 48 h: 1 IC50 = 25.43 lmol Pt L ) displayed higher growth inhibition at the same concentration. These data suggested that conjugation of LHRH could enhance cytotoxicity of the CDDP-loaded polysaccharide nanoparticles in cells overexpressing the LHRH receptor likely due to the high cellular uptake of CDDP, consistent with the quantitative analyses by ICP-MS. 3.2. In vivo toxicity and tolerability

2.10. Statistical analysis All experiments were performed at least 3 times and expressed as means ± standard deviation. Data were analyzed for statistical significance using Student’s test. p < 0.05 was considered statistically significant, and p < 0.01 was considered highly significant.

3. Results and discussion 3.1. Cellular uptake and in vitro cytotoxicity Dex-SA-CDDP is polysaccharide nanoparticle tethered with CDDP via chelate interactions between the ionic polymeric carrier and CDDP (Scheme 1A). Dex-SA-CDDP-LHRH, the targeted counterpart, has similar physicochemical properties. Our previous study has demonstrated that, the Dex-SA-CDDP-LHRH nanoparticles could enhance the cellular internalization in LHRH receptor-positive MCF-7 tumor cells [15]. Based on the previous report that LHRH receptors are overexpressed in 4T1 breast tumor cells [22], the total platinum accumulation in the cells after their treatment with free CDDP and CDDP-loaded nanoparticles were quantitatively assessed. As shown in Fig. 1A, the intracellular platinum content was significantly higher for Dex-SA-CDDP-LHRH (8.91 ± 0.85 ng Pt per 106 cells) than that of Dex-SA-CDDP (5.74 ± 0.63 ng Pt per 106 cells, ⁄⁄p < 0.01) after 5 h incubation. Competitive inhibition on the cellular uptake with free LHRH was further used to confirm whether the uptake of Dex-SACDDP-LHRH was mediated by the ligandreceptor interaction. It was obvious that the addition of free LHRH decreased the uptake of Dex-SA-CDDP-LHRH (5.99 ± 0.62 ng Pt per 106 cells, ⁄⁄p < 0.01), demonstrating that the LHRH conjugated nanoparticles were recognized by LHRH receptors on the surface of 4T1 cells and internalized through receptor-mediated endocytosis [15,22,23]. Nevertheless, it should be noted that the accumulation of free CDDP (12.42 ± 1.25 ng Pt per 106 cells) exceeded that of CDDP-loaded nanoparticles, including the targeted formulation. This result was

Although CDDP is one of the most widely used and effective anticancer drugs, the broader therapeutic applications of CDDP are limited by its severe side effects, such as nephrotoxicity, peripheral neuropathy, nausea, anemia and ototoxicity [26–28]. According to the delayed drug release behavior of Dex-SA-CDDP and Dex-SA-CDDP-LHRH, we proposed that the systemic toxicity of CDDP could be significantly decreased, after its incorporation into the polysaccharide nanoparticles. To evaluate the toxicity and tolerability, the maximum tolerated dose for a single intravenous administration of Dex-SA-CDDP and Dex-SA-CDDP-LHRH was assessed in Kunming mice and compared to free CDDP. The body weight and survival details of the mice were monitored for 10 d after injection of free CDDP at doses of 1, 4, 10, 15, 20 and 25 mg kg1, and Dex-SA-CDDP and Dex-SACDDP-LHRH at doses of 4, 10, 20, 30, 40 and 50 mg kg1 CDDP equivalents (Fig. 2 and Table 1). As shown in Fig. 2A and B, free CDDP was well tolerated at the dose of 1 mg kg1. However, increasing the CDDP dose to 4 mg kg1 resulted in sustained decrease of body weight in the first 4 days and gradual recovery thereafter. Further increasing the dose to 10 mg kg1 would lead to continuous decrease of body weight, and all the mice died within 5 days. A significant body weight loss was observed at 15, 20, and 25 mg kg1 of free CDDP, and all the mice died within 4 day post-injection. For the mice treated with Dex-SA-CDDP and DexSA-CDDP-LHRH, there were no significant body weight loss and noticeable changes in normal activity at a CDDP dose of 4, 10 and 20 mg kg1 (Fig. 2C–F). Further increase of the CDDP dose to 30 mg kg1 resulted in a moderate decrease of body weight. Nevertheless, a gradual recovery of body weight was observed for both Dex-SA-CDDP and Dex-SA-CDDP-LHRH, with the final per cent of body weight at 95.0% and 91.2%, respectively (Fig. 2D and F and Table 1). However, the Dex-SA-CDDP and Dex-SACDDP-LHRH dose above 40 mg kg1 caused animal death (Fig. 2C and E). The maximum tolerated dose was estimated based on the threshold at which all animals survived and the body weight loss was below 20% [29]. The corresponding maximum tolerated dose

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Fig. 1. (A) Platinum uptake by the 4T1 breast cancer cells after incubation of free CDDP, Dex-SA-CDDP, and Dex-SA-CDDP-LHRH for 5 h. For the LHRH receptor blocking study, 4T1 cancer cells were first incubated with 20 lM LHRH for 1 h, followed by co-incubation with Dex-SA-CDDP-LHRH. (B) In vitro cytotoxicity of Dex-SA to 4T1 cells after incubation for 48 h. (C and D) Cytotoxicities of CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH to 4T1 cells after incubation for (C) 24 h and (D) 48 h.

of free CDDP was determined to be 4 mg kg1, which was in accordance with the previous study [30]. Dex-SA-CDDP and Dex-SACDDP-LHRH nanoparticles could increase the maximum tolerated dose of CDDP from 4 to 30 mg kg1, which was likely due to the slow release kinetics for CDDP and decreased nonselective uptake by kidney (Fig. 3) [31]. The significantly improved safety of the CDDP-loaded polysaccharide nanoparticles might allow a full dose of chemotherapy to achieve maximal therapeutic effect. 3.3. Biodistribution study Generally, the xenograft models, such as nude mice bearing human tumor models, are limited in the ability to properly represent the metastatic nature of tumors in the clinic because the tumor is mostly confined in the primary inoculation site [32]. The 4T1 aggressive metastatic breast cancer cell line derived from a Balb/c mouse mammary carcinoma, is a well-established metastatic model [33,34]. In addition, its high expression of LHRH receptors and lymph node-, lung- and liver-specific tumor metastasis has been demonstrated in the previous studies [35,36]. Therefore, it is a desirable tumor model for our research. The biodistribution characteristics of free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH were evaluated in both normal and tumor-bearing Balb/c mice (Fig. 3). Firstly, in vivo biodistribution in normal mice was determined within various mouse organ tissues including, heart, liver, spleen, lung and kidney (Fig. 3A). Free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH at an equivalent CDDP dose of 5 mg kg1 were intravenously injected to each mouse. Tissue samples were taken at 2, 10, and 24 h post injection, and quantified the platinum concentrations using ICP-MS. It is evident that both

free CDDP and the CDDP-loaded nanoparticles dispersed quickly and broadly to all the tested tissues within 2 h post injection. As shown in Fig. 3A, matching with the previous data, free CDDP quickly diffused into and cleared out from each organ, while a significant fast and high distribution was observed in the kidney [19,37]. The nephrotoxicity of CDDP was dependent on the maximum platinum concentration in the kidney tubule, therefore, the high peak concentration of platinum in the kidney would induce severe nephrotoxicity [38]. In contrast, both Dex-SA-CDDP and Dex-SACDDP-LHRH could effectively reduce the accumulation of CDDP in kidney so as to reduce the acute nephrotoxicity of CDDP. The accumulation of platinum in the kidney 2 h following Dex-SA-CDDP and Dex-SA-CDDP-LHRH delivery was 58.8% (2963.7 ± 463.3 ng g1) and 56.9% (2867.5 ± 635.5 ng g1) of that of free CDDP (5038.4 ± 740.7 ng g1), respectively. On the other hand, significantly greater platinum accumulation was seen in the liver, spleen and lung of Dex-SA-CDDP and Dex-SA-CDDP-LHRH nanoparticle-treated mice than in animals treated with free CDDP, especially at 24 h post injection. After 24 h, the platinum concentrations in the liver after Dex-SA-CDDP and Dex-SA-CDDP-LHRH treatment were 1.80-fold (5161.8 ± 854.7 ng g1) and 1.83-fold 1 (5246.8 ± 846.1 ng g ) respectively, greater than those of free CDDP (2873.3 ± 956.9 ng g1). The platinum concentrations in the spleen and lung after Dex-SA-CDDP (spleen: 1.86-fold, 2062.9 ± 620.5 ng g1; lung: 1.55-fold, 2080.0 ± 533.4 ng g1) and Dex-SA-CDDP-LHRH (spleen: 1.83-fold, 2032.0 ± 460.7 ng g1; lung: 1.47-fold, 1973.7 ± 388.9 ng g1) treatment were greater than those of free CDDP (spleen: 1108.9 ± 204.3 ng g1; lung: 1341.7 ± 265.3 ng g1). The above results indicated that most of the free CDDP was eliminated through glomerular filtration, while

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Fig. 2. (A, C and E) Survival rate and (B, D and F) body weight change of Kunming mice treated with (A and B) free CDDP at a dose of 1, 4, 10, 15, 20 and 25 mg kg1, (C and D) Dex-SA-CDDP and (E and F) Dex-SA-CDDP-LHRH at an equivalent CDDP dose of 4, 10, 20, 30, 40 and 50 mg kg1.

the Dex-SA-CDDP and Dex-SA-CDDP-LHRH nanoparticles were taken up by the reticuloendothelial system (RES) after their systemic administration [26]. There were no significant differences in platinum concentrations in the heart tissues among free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH groups after 24 h. Next, the biodistribution study in 4T1 tumor-bearing mice was carried out to confirm the specificity of the targeted polysaccharide nanoparticles. When the tumor volumes reached approximately 400 mm3 and significant metastatic foci were detected in the liver and lung by H&E staining, the mice were administered intravenously with free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH nanoparticles. Heart, liver, spleen, lung, kidney and tumor were excised at defined time intervals and the platinum content was measured by ICP-MS after treating by nitric acid. As shown in Fig. 3B, both Dex-SA-CDDP and Dex-SA-CDDP-LHRH nanoparticles showed higher drug concentrations in tumor than free CDDP at all

the time points examined, which could be contributed to the prolonged blood circulation, delayed drug release and excellent passive targeting effective of the nanosized drug delivery system [39]. Importantly, Dex-SA-CDDP-LHRH exhibited a higher drug concentration as compared to Dex-SA-CDDP, demonstrating the specific tumor targeting effect of LHRH peptide. After 24 h, the targeted Dex-SA-CDDP-LHRH nanoparticles induced greater platinum tumor accumulation (3376.8 ± 763.0 ng g1) than those of the nontargeted Dex-SA-CDDP nanoparticles (2137.4 ± 332.2 ng g1), and Dex-SA-CDDP delivered significantly higher levels of platinum to the tumor than free CDDP (1265.8 ± 334.7 ng g1). Similar distribution characteristics of free CDDP, Dex-SA-CDDP and DexSA-CDDP-LHRH were observed in heart and kidney. However, the CDDP-loaded nanoparticles, especially the Dex-SA-CDDP-LHRH exhibited an increased drug concentration in the lung, liver and spleen of the 4T1 tumor-bearing mice, as compared to the

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Table 1 Dosing information of free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH for maximum tolerated dose studies in Kunming mice. Groups

Dose Number Number Mean (mg kg1) of mice of death survival time (day)

Per cent of body weight at day 10 (%)

Free CDDP

1 4 10 15 20 25

3 3 3 3 3 3

0 0 3 3 3 3

10.0 10.0 4.3 ± 1.2 2.7 ± 1.5 2.0 ± 1.0 1.7 ± 0.6

122.0 ± 5.6 102.8 ± 5.1    

Dex-SA-CDDP

4 10 20 30 40 50

3 3 3 3 3 3

0 0 0 0 1 3

10.0 10.0 10.0 10.0 7.7 ± 4.0 3.7 ± 1.2

119.9 ± 4.2 116.0 ± 9.4 106.6 ± 4.5 95.0 ± 5.1 75.5 ± 5.0 

4 10 20 30 40 50

3 3 3 3 3 3

0 0 0 0 2 3

10.0 10.0 10.0 10.0 6.0 ± 3.6 3.3 ± 0.6

116.4 ± 6.0 113.3 ± 5.3 104.0 ± 7.8 91.2 ± 7.5 75.7 

Dex-SA-CDDP-LHRH

platinum concentration in the corresponding organs of normal mice. Moreover, the platinum levels in these organs were significantly higher in tumor-bearing mice treated with the targeted Dex-SA-CDDP-LHRH nanoparticles than those with the non-targeted Dex-SA-CDDP nanoparticles, while there was no significant difference between the Dex-SA-CDDP-LHRH and Dex-SA-CDDP groups in normal mice. Based on the above results, it could be concluded that: (1) the CDDP-loaded nanoparticles could significantly decrease the drug accumulation in kidney, thus reduce the nephrotoxicity of CDDP and improve the tolerability of small molecule drug; (2) both Dex-SA-CDDP and Dex-SA-CDDP-LHRH nanoparticles improved the drug accumulation in tumor by prolonged blood circulation

and the EPR effect; (3) the targeted Dex-SA-CDDP-LHRH nanoparticles not only increased platinum accumulation in the injected primary tumor but also the metastasis-containing organs, so that they suppressed the tumor growth and inhibited its metastasis simultaneously; (4) although the nanomedicines could not avoid RES and resulted in increased accumulation in liver and spleen, the delayed drug release behavior and fast regeneration with healthy hepatocyte and splenocyte could help the animals for effective recovery from the metabolic break [40].

3.4. In vivo anticancer efficacy The antitumor activity and systemic toxicity of the CDDP-loaded nanoparticles were evaluated in 4T1 orthotopic breast tumor model. Mice bearing 4T1 tumors were treated with PBS, free CDDP (2.0 mg kg1), free CDDP (5.0 mg kg1), Dex-SA-CDDP (5.0 mg kg1 on CDDP basis), Dex-SA-CDDP (10.0 mg kg1 on CDDP basis), Dex-SA-CDDP-LHRH (5.0 mg kg1 on CDDP basis) and Dex-SA-CDDP-LHRH (10.0 mg kg1 on CDDP basis), respectively, by intravenous injection on days 0, 4, and 8. As shown in Fig. 4A, the tumors in all drug-treated groups showed growth retardation in a dose-dependent manner compared to the PBS control. Free CDDP at a low dose of 2 mg kg1 could not effectively inhibit the tumor growth. Increasing the CDDP dose to 5 mg kg1 would contribute to the antitumor efficacy, however, all the mice died within 8 days because of the acute toxicity. All the mice treated with the CDDP-loaded nanoparticles at a dose of 5 and 10 mg kg1 were alive during the experimental period, suggesting the improved safety and tolerance. Table 2 shows the mean tumor volume at day 20 of the mice treated with different formulations. Compared with the group treated with PBS (1367 ± 209 mm3) as the control, free CDDP at a dose of 2 mg kg1 (912 ± 133 mm3, ⁄⁄ p < 0.01) only showed moderate antitumor efficacy. In contrast, both the targeted Dex-SA-CDDP-LHRH (5 mg kg1: 613 ± 90 mm3, ⁄⁄⁄ p < 0.001; 10 mg kg1: 370 ± 64 mm3, ⁄⁄⁄p < 0.001) and the nontargeted Dex-SA-CDDP (5 mg kg1: 752 ± 106 mm3, ⁄⁄⁄p < 0.001;

Fig. 3. Biodistribution studies of free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH in (A) normal Balb/c mice and (B) 4T1 tumor-bearing mice. All the mice were injected via tail vein at an equivalent CDDP dose of 5.0 mg kg1. Selected tissues were harvested at different time points, weighted and measured for the platinum content by ICP-MS. Data were shown as means ± standard deviation. (n = 3).

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Fig. 4. In vivo tumor growth inhibition of free CDDP, Dex-SA-CDDP and Dex-SA-CDDP-LHRH in 4T1 orthotopic mammary tumor model. (A) The tumor volumes of mice as a function of time. Data were presented as means ± standard deviation (n = 6). The arrows represented the day on which the intravenous tail vein injection was performed. (B) Body weights of the mice over the experimental period. (C) The tumor weight of each group at the end of the experiment. (D) Tumor inhibition rates of various formulations.

Table 2 In vivo antitumor effects of different formulations in orthotopic mammary tumor model (n = 6). Groups

PBS Free CDDP (2 mg kg1) Dex-SA-CDDP (5 mg kg1) Dex-SA-CDDP (10 mg kg1) Dex-SA-CDDP-LHRH (5 mg kg1) Dex-SA-CDDP-LHRH (10 mg kg1)

Mean tumor volume at day 20 (mm3)

Compare with PBS

Free CDDP (2 mg kg1)

Dex-SA-CDDP (5 mg kg1)

Dex-SA-CDDP (10 mg kg1)

Dex-SA-CDDP-LHRH (5 mg kg1)

1367 ± 209 912 ± 133 752 ± 106 467 ± 79 613 ± 90

– p < 0.01 p < 0.001 p < 0.001 p < 0.001

– – p < 0.05 p < 0.001 p < 0.01

– – – p < 0.001 p < 0.05

– – – – p < 0.05

– – – – –

p < 0.001 p < 0.001

p < 0.001

p < 0.05

p < 0.001

370 ± 64

10 mg kg1: 467 ± 79 mm3, ⁄⁄⁄p < 0.001) exhibited obvious inhibition, especially at the high dose of 10 mg kg1. Moreover, at the same CDDP dose, the LHRH modified nanoparticles displayed a superior therapeutic effect compared with the non-modified nanoparticles, which could be contributed to the higher accumulation in the tumor site, as proven in the in vivo biodistribution study. Tumor weight was measured at the termination of the experiment and tumor inhibition ratios were calculated (Fig. 4C and D). The tumor inhibition ratios calculated from tumor weight were in good consistency with the results from tumor volume measurements. These results also demonstrated that the targeted Dex-SA-CDDP-LHRH nanoparticles could improve the therapeutic effect as compared to its non-targeted counterpart.

The body weights of the treated mice were monitored throughout the experiment and used as an indication to investigate the adverse effects of different drug formulations (Fig. 4B). Mice receiving free CDDP at a high dose of 5 mg kg1 showed a significant loss of body weight, with about 25% body weight loss on day 8 after treatment. Even at a low CDDP dose of 2 mg kg1, the acute toxicity of mice was obvious, as 17% loss of their initial weight was observed on day 20 (⁄⁄⁄p < 0.001, compared with PBS and all the CDDP-loaded nanoparticles groups). In contrast, mice treated with the Dex-SA-CDDP and Dex-SA-CDDP-LHRH nanoparticles only resulted in about 5% body weight loss even at a high dose of 10 mg kg1. These results demonstrated that no noticeable systemic toxicity was caused by the two doses of the

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CDDP-loaded polysaccharide nanoparticles throughout the treatment period. 3.5. Histological and immunohistochemical analyses To further investigate the antitumor efficacy, the tumors treated with various formulations were dissected and sectioned for pathology analysis at the termination of the trials. As shown in Fig. 5A, the tumor sections treated with PBS were composed of tightly packed tumor cells interspersed with large amount of stroma. The tumor cells with a large nucleus and a spherical or spindle shape were observed, in which more chromatin and binucleolates were also observed, indicating a rapid tumor growth [41]. However, after the treatment with chemotherapeutic agents, the histological features of tumors exhibited a significant difference from the control group. The tumor cellularity decreased significantly and various degrees of tissue necrosis, extensive nuclear shrinkage and fragmentation were observed in all the drug-treated groups. Additionally, many of the tumor cells were composed of membrane-bound, small nuclear fragments surrounded with a rim of cytoplasm, exhibiting typical apoptotic characteristics

[42]. Increasing the drug dose of CDDP-loaded nanoparticles would significantly improve the antiproliferation effect. At the same CDDP dose, the targeted Dex-SA-CDDP-LHRH nanoparticles obviously enhanced the inhibition of tumor cell proliferation, as compared with the non-targeted formulation. Especially for the Dex-SA-CDDP-LHRH at a high dose of 10 mg kg1, chromatin was concentrated and distributed around the edge, and nuclei became pyknotic, fragmented or absent, and the necrosis area was the largest among the tested groups. This was also consistent with the in vivo antitumor capability that the Dex-SA-CDDP-LHRH group (10 mg kg1) exhibited the highest antitumor efficacy. The immunohistochemistry analysis of the tumor sections was further conducted to detect the expression of cleaved 25 kDa fragment of PARP1 (c-PARP1), one of the essential substrates cleaved by both caspase-3 and -7 and a characteristic hallmark of apoptosis [20,43]. Compared with PBS or free CDDP, administration of the CDDP-loaded nanoparticles markedly increased the number of cPARP1-positive signals, indicating that more cells underwent apoptosis in these groups. All these results were consistent with the data of inhibition of tumor growth and further confirmed that the actively targeted Dex-SA-CDDP-LHRH nanoparticles could

Fig. 5. Ex vivo histological and immunohistochemical analyses of tumor, kidney, lymph node, lung and liver sections of mice treated with (a) PBS, (b) free CDDP (2.0 mg kg1), (c) Dex-SA-CDDP (5.0 mg kg1 on CDDP basis), (d) Dex-SA-CDDP (10.0 mg kg1 on CDDP basis), (e) Dex-SA-CDDP-LHRH (5.0 mg kg1 on CDDP basis) and (f) Dex-SA-CDDPLHRH (10.0 mg kg1 on CDDP basis). (A) H&E and immunohistochemical analyses of 4T1 tumor sections (20 days after the first treatment). Nuclei were stained bluish violet, whereas extracellular matrix and cytoplasm were stained pink in H&E staining. Brown and blue stains indicated cleaved PARP1 and nuclei, respectively, in immunohistochemical assay. (B) Representative H&E sections of the kidney, lymph node, lung and liver from the control and drug-treated mice. The dashed yellow lines indicated the metastatic foci. The green arrows pointed to the normal lung tissues. The blue arrows indicated the hepatic veins. The scale bars represent 100 lm.

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Fig. 6. Therapeutic monitoring of late-stage 4T1 tumor metastasis after the treatment of (a) PBS, (b) free CDDP (3.0 mg kg1), (c) Dex-SA-CDDP (10.0 mg kg1 on CDDP basis), and (d) Dex-SA-CDDP-LHRH (10.0 mg kg1 on CDDP basis). (A) Experimental schedule for tumor induction and drug treatments. (B) The picture of the sentinel lymph node in different groups at the end of the experiment. (C) The lymph node weight of each group. (D) Representative H&E sections of the lymph node, lung and liver. The scale bars represent 100 lm. The dashed yellow lines and yellow arrows indicated the metastasis foci.

efficiently deliver CDDP to the tumor site and persistently inhibit the growth of 4T1 breast cancer. Although CDDP has been a mainstay for chemotherapy, its clinical use is often restricted by the following side-effects, especially the dose-dependent nephrotoxicity, which negatively affects

patients’ quality of life [44]. Thus, new drug formulations, which could protect the kidney from CDDP damage without compromising its antitumor activity, would be of great clinical benefit. To this end, the nephrotoxicity of free CDDP and the CDDP-loaded nanoparticles was studied. As shown in Fig. 5B, free CDDP group showed severe

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renal injury, including marked necrosis in proximal tubules, thickening of the mesangium and glomerular basement membrane, loss of brush border in renal tubules, and contraction of epithelial luminal space. Whereas, the nephrotoxicity of CDDP was significantly inhibited after chelation with Dex-SA, suggesting that the CDDPloaded polysaccharide nanoparticles could allow the long-term administration due to the controlled release behavior. Sentinel lymph node is the hypothetical first lymph node or group of nodes reached by metastasizing cancer cells traveling through lymphatic vessels from a primary tumor [45,46]. Evaluating the nodal status is very important for diagnosis of cancer progression and metastasis, and accordingly determines prognosis and the appropriate therapeutic schedule [47,48]. For example, most of the patients with breast cancer and melanoma are currently subject to invasive sentinel lymph node biopsy to stage metastases [48,49]. In our mammary tumor metastasis model, a large number of metastatic sites (marked by dashed yellow lines) in the nearby sentinel lymph node of untreated mice were observed at day 20 (Fig. 5B). Aggressive lymph node metastases were also observed in mice treated with free CDDP, indicating that the treatment with free CDDP at a low dose was not sufficient to prevent cancer metastasis. In contrast, compared to PBS- and free CDDP-treated groups, only minimal signal of lymph node metastasis was observed in mice treated with CDDP-loaded nanoparticles, especially at a high drug dose. Moreover, the targeted nanoparticles could further inhibit the lymph node metastasis. Besides lymph node metastasis, 4T1 cells are documented to be very aggressive and primary tumor that has been established in Balb/c mouse typically metastasizes to the lung, and liver [50]. As shown in Fig. 5B, the mice treated with PBS had massive tumor metastases in lung, with the majority of the organic tissues occupied by tumor cells. Moreover, for mice administrated with PBS and free CDDP, a focal of the tumor metastasis was observed in liver tissues due to the diffuse metastatic lesion consisted of neoplastic cells around the hepatic vessels. In contrast, for the mice treated with CDDP-loaded nanoparticles, the areas of metastatic lesion in lung and liver decreased in a dose-dependent manner. Generally, administration of the LHRH modified nanoparticles markedly inhibited spontaneous lung and liver metastases as compared with the non-targeted nanoparticles and free CDDP, exhibiting only minimal levels of metastasis in these organs, and the architectures were left largely intact. Similar to the previous studies [19,32,33], our results also demonstrated that the antimetastasis efficacy was consistent with the primary tumor growth retardation effect (Figs. 4A and 5B). The efficient inhibition of tumor metastasis might also benefit from the significant retardation of primary tumor growth, because larger tumors could potentially promote more metastasis due to their increased size and/or altered microenvironment [51]. To further investigate the antimetastasis efficacy of Dex-SA-CDDP-LHRH on metastatic 4T1 tumor in late stage, the treatments were started when tumors grew to a volume of 400–500 mm3. In most cases, the size of lymph nodes was indicative of the presence of metastases. Macroscopically, regional lymph nodes were enlarged when the tumor cells metastasize to the lymph nodes [35]. As shown in Fig. 6B and C, compared with the control group, the enlargement of lymph node was effectively inhibited in all the chemotherapy groups. As compared to the non-targeted nanoparticles (Dex-SA-CDDP, group c), intravenous injection of the targeted nanoparticles (Dex-SA-CDDP-LHRH, group d) was more efficient in inhibiting lymph node enlargement. Moreover, as revealed by the H&E stained slices, Dex-SA-CDDP-LHRH nanoparticles significantly suppressed the metastasis of late-stage 4T1 tumor to lymph node, lung and liver (Fig. 6D). Taken together, the present study demonstrated that Dex-SA-CDDP-LHRH

nanoparticles have advisable potential for antimetastasis therapy in both early- and late-stage metastatic breast cancers. The LHRH modification increased the antimetastasis effect probably because of the specific targeting to the primary tumor cells and metastatic cells in the specific organs, as well as the cells spreading in the blood or lymphatic routes [32]. All the experimental data have clearly evidenced that the Dex-SA-CDDP-LHRH treatment afforded significant advantages in inhibiting 4T1 breast tumor metastasis, which was an important advance in clinical cancer therapy. 4. Conclusion In summary, the CDDP-loaded LHRH-modified dextran nanoparticles were used as the targeted nanomedicine to suppress murine breast cancer growth and metastasis. The Dex-SA-CDDP-LHRH nanoparticles increased the cellular uptake and cytotoxicity in vitro in 4T1 cell line, compared with the non-targeted counterparts. Both the non-targeted and targeted nanoparticles could significantly decrease the systemic toxicity of CDDP and increase the maximum tolerated dose of CDDP from 4 to 30 mg kg1. More importantly, the LHRH-targeted nanoparticles led to significantly higher accumulation of CDDP in the injected primary tumor and metastasis-containing organs, and meanwhile significantly reduced the nephrotoxicity of CDDP. Desirable antitumor effect and antimetastasis efficacy of Dex-SA-CDDP-LHRH were confirmed in the Balb/c mice bearing 4T1 tumors with low systemic toxicity, indicating that the LHRHdecorated nanoparticles showed great potential for the targeted chemotherapy of metastatic breast cancer. In addition, our strategy can be extended to other types of platinum-based chemotherapeutic agents and targeting ligands for the treatment of other types of malignant tumors. Acknowledgments This research was financially supported by National Natural Science Foundation of China (Projects 51173184, 51373168, 51390484, 51233004 and 51321062), Ministry of Science and Technology of China (International Cooperation and Communication Program 2011DFR51090) and Program of Scientific Development of Jilin Province (20130727050YY and 20130206066GX). The authors have declared no conflict of interest. Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Figs. 2–6 and Scheme 1 are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10. 1016/j.actbio.2015.02.022. References [1] Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64:9–29. [2] Murakami M, Ernsting MJ, Undzys E, Holwell N, Foltz WD, Li SD. Docetaxel conjugate nanoparticles that target alpha-smooth muscle actin-expressing stromal cells suppress breast cancer metastasis. Cancer Res 2013;73:4862–71. [3] Shen J, Sun H, Xu P, Yin Q, Zhang Z, Wang S, et al. Simultaneous inhibition of metastasis and growth of breast cancer by co-delivery of twist shRNA and paclitaxel using pluronic P85-PEI/TPGS complex nanoparticles. Biomaterials 2013;34:1581–90. [4] Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2002;2:563–72. [5] He Q, Shi J. MSN anti-cancer nanomedicines: chemotherapy enhancement, overcoming of drug resistance, and metastasis inhibition. Adv Mater 2014;26:391–411. [6] Xiao J, Duan X, Meng Q, Yin Q, Zhang Z, Yu H, et al. Effective delivery of p65 shRNA by optimized Tween 85-polyethyleneimine conjugate for inhibition of tumor growth and lymphatic metastasis. Acta Biomater 2014;10:2674–83.

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