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Epidermal Growth Factor Receptor Targeting Peptide Nanoparticles Simultaneously Deliver Gemcitabine and Olaparib to Treat Pancreatic Cancer with Breast Cancer 2 (BRCA2) Mutation Chong Du, Yingqiu Qi, Yinlong Zhang, Yazhou Wang, Xiao Zhao, Huan Min, Xuexiang Han, Jiayan Lang, Hao Qin, Quanwei Shi, Zhengkui Zhang, Xiaodong Tian, Greg J Anderson, Ying Zhao, Guangjun Nie, and Yinmo Yang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01573 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 8, 2018

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Epidermal Growth Factor Receptor Targeting Peptide Nanoparticles Simultaneously Deliver Gemcitabine and Olaparib to Treat Pancreatic Cancer with Breast Cancer 2 (BRCA2) Mutation

Chong Du1,2‡, Yingqiu Qi2,3‡, Yinlong Zhang2,4, Yazhou Wang1,2, Xiao Zhao2, Huan Min2,3, Xuexiang Han2,6, Jiayan Lang2,6, Hao Qin2,6, Quanwei Shi2,6, Zhengkui Zhang1, Xiaodong Tian1, Greg J Anderson5, Ying Zhao2,6*, Guangjun Nie2,6* and Yinmo Yang1* 1Department

of General Surgery, Peking University First Hospital, Beijing 100034,

China 2CAS

Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China 3College

of Science, Northeastern University, Shenyang 110819, China

4College

of Pharmaceutical Science, Jilin University, Changchun 130021, China

5QIMR

Berghofer Medical Research Institute, Royal Brisbane Hospital, QLD 4029,

Australia 6University

of Chinese Academy of Sciences, Beijing 100049, China

‡These authors contributed equally to this work. *Correspondence to: [email protected] or [email protected] [email protected]

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ABSTRACT Pancreatic cancer (PCa) is one of the most lethal malignancies with 5-year survival rate of less than 8%. Current treatment regiments have a low response rate in unselected patients. However, the subgroup of PCa patients with BRCA mutations may benefit from poly-ADP-ribose polymerase inhibitors (PARPi) due to their biological properties in DNA repair. Dose-limiting toxicity in normal tissues is frequently observed when PARPi are combined with other chemotherapies, and the co-delivery of two drugs to tumor sites at an adequate concentration is challenging. To address this issue, we have engineered an epidermal growth factor receptor (EGFR) targeting (with GE11 peptide) self-assembly amphiphilic peptide nanoparticle (GENP) to co-deliver gemcitabine and the PARPi olaparib to treat BRCA mutant PCa. The GENP was relatively stable, exhibited high encapsulation efficiency, and could coordinately release the two drugs in tumor milieu. Gemcitabine and olaparib showed strong synergistic actions in optimized conditions in vitro. The nanoparticle prolonged the half-life of both drugs and resulted in their tumor accumulation at the optimal therapeutic ratio in vivo. The drug-loaded nanoparticles were able to significantly suppress tumor growth in a murine PCa model with minimal side effects. Drug co-delivery of DNA damaging agents and PARP inhibitors via the GENP represents a promising approach for treatment of pancreatic cancers with molecular defects at DNA repair pathway.

KEYWORDS: pancreatic cancer, BRCA2 mutation, synthetic lethality, drug co-delivery, self-assembly peptide nanoparticle


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Pancreatic cancer (PCa) is one of the leading causes of cancer death in the world.1, 2 It has a median survival of 6 months and a 5 years survival rate that remains less than 8%.2 The standard first-line chemotherapy for PCa, gemcitabine (Gem), has been associated with low response rates and has proven only modestly effective in unselected patient populations.3 A subset of PCa patients carries mutations in the BRCA tumor suppressor genes.4 BRCA1 and BRCA2 are involved in the repair of DNA double-strand breaks (DSBs) through the homologous recombination (HR) pathway.5 Estimates of BRCA gene mutation prevalence in sporadic pancreatic cancer have ranged from 5.5 to 21.6%.6-8 The biological properties of this subgroup means that they may potentially benefit from therapeutic agents targeting DNA repair pathways.9, 10 Poly-ADP-ribose polymerase (PARP) plays a key role in the repair of single-strand DNA breaks (SSBs).11 Its inhibition is thought to result in the accumulation of SSBs in DNA, and this eventually can lead to DSBs.12 Therefore, PARP inhibitors (PARPi) are synthetically lethal in the presence of HR deficiency as inhibition of PARP leads to the persistence of DNA lesions that would normally be repaired by BRCA-mediated HR.13 The FDA has approved a PARP inhibitor olaparib (Ola, Lynparza) to treat patients with BRCA-mutated advanced ovarian cancer14 and breast cancer,15 and it has considerable potential in the treatment of other BRCA1/2 mutant tumors. The combination therapy of Gem and Ola may benefit PCa patients with BRCA mutations. A key aspect of drug combination therapy is ensuring that appropriate concentration of each drug reach the target tissue. Dose-limiting normal tissue toxicity has frequently been observed when a PARPi has been used with other chemotherapies in the clinic.16 One of the main reasons for this is that relative large amounts of drug must be administered systemically to overcome the obstacles of poor plasma stability, low tissue perfusion, and inefficient cellular uptake. This comes at the expense of toxicity. Considering their merits of prolonged circulation time, enhanced tissue accumulation and ability to facilitate effective co-delivery, using a nanocarrier to deliver a combined drug regime has considerable appeal.17, 18 To date, more than 150


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nanocarrier-based cancer therapeutic drugs are in various stages of development.19, 20 Moreover, some drugs are widely used in the clinic, such as Abraxane (albumin-bound paclitaxel), which extend the survival of pancreatic cancer patients by several months when co-administered with Gem.21 Due to their chemical versatility, and ability to specifically recognize other biological macromolecules,22 peptides and peptide derivatives have been widely used for multifunctional nanostructure in drug delivery. We have previously shown that self-assembly amphiphilic peptides provide attractive vehicles for delivering chemotherapeutic drugs to tumors.23-26 In this study, we report an amphiphilic peptide nanocarrier that actively targets epidermal growth factor receptor (EGFR), which is overexpressed on the surface of PCa cells, to co-deliver Gem and Ola to treat BRCA2 mutant pancreatic cancer. Gem and Ola acted synergistically in vitro to enhance the killing of BRCA2 mutant PCa capan-1 cells, and this ability was retained when they were incorporated into nanoparticles. Moreover, the nanoparticles prolonged the half-life of both drugs and resulted in their accumulation at the tumor site at the optimal therapeutic ratio. Finally, in pancreatic tumor bearing mice, drug-loaded nanoparticles targeted strongly to the tumors and were more effective in inhibiting tumor growth than the combination of the free drugs. They achieved this with limited toxic side effects.

RESULTS AND DISCUSSION Preparation and Characterization of Self-assembly Peptide Nanoparticle. To achieve the goal of PCa-cells targeting and drug delivery, an EGFR-targeting peptide (GE11) based amphiphilic peptide (C18-EEG-GE11) monomer was designed and synthesized. Its hydrophilic domain contained GE11 with the amino acid residue sequence YHWYGYTPQNVI that has been verified to hold powerful EGFR-binding efficiency.27,

28

An extra glycine and two glutamic acids containing carboxyl group

were added into the hydrophilic domain to increase its hydrophilicity and to provide negative charges for the hydrophilic head at pH 7.4, in order to decrease nonspecific


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phagocytosis by the reticuloendothelial system (RES) and prolong blood circulation time in vivo. Furthermore, one octadecanoic acid molecule (C18) was linked to be embedded as the hydrophobic tail, which has been well-documented to drive peptide self-assembly (Scheme 1). We also synthesized a non-targeting amphiphilic peptide (C18-EEG-HW12)

with

a

similar

structure

and

scrambled

sequence

of

C18-EEG-GE11 as a control.27

Scheme 1. Proposed mechanism of EGFR targeting self-assembly peptide nanoparticles (GENP) to co-deliver gemcitabine and olaparib to treat BRCA mutant PCa. When PARP is inhibited by olaparib, the SSBs of DNA induced by gemcitabine cannot be repaired via PARP mediated pathway and leads to DSBs. However, the HR pathway for DSB repair is deficient in BRCA mutant pancreatic cancer cells and unrepaired DSBs eventually lead to cell apoptosis. EGFR, epidermal growth factor receptor; ECM, extracellular matrix; PARP, poly ADP-ribose polymerase; HR, homologous recombination; SSBs, single-strand breaks; DSBs, double-strand breaks.

Both amphiphilic peptides C18-EEG-GE11 and C18-EEG-HW12 had low


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critical micelle concentration (CMC) (C18-EEG-GE11, 6.02 M or 12.04 mg/L; C18-EEG-HW12, 7.03 M or 14.06 mg/L) in PBS at pH 7.4, implying that they could readily self-assemble to form core-shell structured peptide nanoparticles (GENP and HWNP) at concentrations equal to or above the CMC, with a hydrophobic octadecanoic acid core and a hydrophilic EGFR-targeting peptide shell towards the surrounding aqueous environment (Figure 1B, Figure S1B). To ensure that the nanoparticles remain stable following dilution both in vitro and in vivo, a concentration that was 60-fold higher than the CMC (approximately 1 mg/mL) was used in the subsequent experiments. At this concentration, transmission electron microscopy (TEM) examination showed that the GENP had a uniform diameter of approximately 30 nm (Figure 1A). When loaded with Gem (GENP-Gem) or Ola (GENP-Ola), the diameters increased to approximately 60 nm, and to approximately 120 nm when both drugs were encapsulated (GENP-Gem-Ola) (Figure 1A, Table S1). HWNP had similar diameters under the same conditions (Figure S1A, Table S1). Dynamic light scattering (DLS) was used to evaluate the nanoparticle size distribution. As shown in Figure S2, the average diameters were approximately 36.66 ± 3.93, 62.18 ± 2.41, 66.70 ± 2.03 and 131.27 ± 3.12 nm for GENP, GENP-Gem, GENP-Ola and GENP-Gem-Ola, respectively. The average diameters were approximately 36.64 ± 2.76, 62.41 ± 3.16, 64.71 ± 4.74 and 121.33 ± 9.33 nm for HWNP, HWNP-Gem, HWNP-Ola and HWNP-Gem-Ola, respectively. Taken together, these results demonstrated that the C18-EEG-GE11 and C18-EEG-HW12 amphiphilic peptides can self-assemble into regular nanostructures and provided the basis for subsequent in vitro and in vivo experiments. High drug loading efficiency and controlled release are important characteristics for nanocarriers as they could improve efficacy and reduce toxicity of the drugs to normal tissues. We showed by high performance liquid chromatography (HPLC) that Gem and Ola could be successfully encapsulated within GENP or HWNP. For individual drug encapsulation efficiency, Gem and Ola have relative high encapasulation efficiency in GENP-Gem/HWNP-Gem and GENP-Ola/HWNP-Ola (Table S2,3). To obtain the optimized ratios of Gem and Ola encapsulated in GENP


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or HWNP, different combinations of peptides and drug ratios have been studied. The results have been summarized in Table S4,5. The in vitro stability of drug loaded nanoparticles were investigated by dispersing the nanoparticles in 10% FBS/PBS (pH 7.4) for 48 h, followed by examination of the changes of morphology by TEM (Figure S3). No significant changes in the morphology of either type of nanoparticles were observed after 48 h incubation, indicating considerable stability of the nanoparticles. Thus, the property of reducing degradation rate of the nanoparticles can afford their cargoes a prolonged circulation time in vivo. We next assessed the drug release profiles of GENP-Gem-Ola and HWNP-Gem-Ola nanoparticles by dialysis against PBS (pH 7.4) at 37°C. Sustained and coordinate release of both Gem and Ola was observed (Figure 1C, Figure S1C). After 12 h of incubation, the cumulative release of each drug reached approximately 50%, and more than 80% was released after 48 h.

Figure 1. Characterization of GENP based self-assembly nanoparticles. (A) Schematic diagram of nanoparticles’ morphology change after drug loading (upper row). The morphology of GENP,


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GENP-Gem, GENP-Ola, and GENP-Gem-Ola was assessed using TEM (lower row). The concentration of peptide in each solution was 1 mg/mL. The scale bar represents 200 nm. (B) Determination of the CMC of the C18-EEG-GE11 peptide. The peptide can form micelles at a concentration equal to or above 6.02 M (12.04 mg/L). (C) Release profiles of Gem and Ola from GENP-Gem-Ola in 10% FBS/PBS over a period of 48 h.

Tumor Cell Targeting in Vitro. To confirm the targeting specificity of the drug-containing nanoparticles, we incubated 1 mg/mL FITC-labeled GENP (GENP-FITC) or HWNP (HWNP-FITC) with human capan-1 PCa cells for 15, 30 or 60 min at 37°C. Western blotting demonstrated that EGFR was highly expressed on the membrane of capan-1 cells, but expressed at low levels on primary human pancreatic stellate cells (H-PSC) and human umbilical vein endothelial cells (HUVEC) (Figure S4). Flow cytometry analysis showed that the binding of GENP-FITC (with the targeting peptide) to capan-1 cells was significantly greater than that of the non-targeted HWNP-FITC (Figure 2A,B). Cellular uptake was studied using confocal laser scanning microscopy (CLSM). Consistent with the flow cytometry analyses, there were extensive internalization of GENP by capan-1 cells but little signal with the untargeted nanoparticles (Figure 2C). In addition, GENP-FITC bound much more strongly to capan-1 cells than to H-PSC or HUVECs which have lower EGFR levels (Figure S5). These data suggest that GENP actively target cells highly expression of EGFR, such as PCa cells.


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Figure 2. Cellular uptake of FITC labeled nanoparticles. (A) Representative fluorescence intensity measured by flow cytometry for FITC in capan-1 cells treated with control medium, non-targeted HWNP or targeted GENP for 15, 30 and 60 min at 37°C. (B) Comparison of the mean fluorescent intensity between different groups shown in (A) at each time point. *** p < 0.001. (C) Representative confocal microscopic images of capan-1 cells treated with GENP-FITC or HWNP-FITC at different time points. Cell nuclei (blue) were stained with Hoechst 33342, and FITC appears in green. Scale bar = 20 m.

Synergistic Inhibitory Effects of Gemcitabine and Olaparib in BRCA2 Mutant Capan-1 Cells. As confirmed in Figure S6, capan-1 cells harbor a 6174delT mutant BRCA2 allele, which results in expression of truncated BRCA2 protein with a deficiency in homologous recombination. To demonstrate whether Gem and Ola could act synergistically in vitro, we initially established the IC50 of free Gem and Ola in capan-1 cells, obtaining values of 36 ng/mL and 1.52 g/mL, respectively (Figure S7). We next examined the effects of a mixture of free Gem and Ola (Gem+Ola), and Gem and Ola encapsulated within GENP (GENP-Gem-Ola) on


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capan-1 cells. A series of Gem+Ola mixtures was prepared using a fixed amount of Gem plus different amounts of Ola to yield Gem/Ola ratios over the range of 1:1 to 1:100. We used each mixture as a starting concentration from which a series of dilutions was made. These samples were used to conduct cell viability experiments, thus we can study the interactions between free Gem and Ola by utilizing CompuSyn software.29 The combination index (CI) of Gem combined with Ola at different ratios was calculated (Figure S8). An antagonistic effect was represented by a CI > 1, and additive or weak synergistic effect at 0.5 < CI ≤ 1 and a strong synergistic effect if CI < 0.5. As shown in Figure S8, a strong synergistic effect was observed when the drug ratio was 1:10. Consistent with the results using free drugs, GENP-Gem-Ola exhibited a strong synergy at a 1:10 ratio (Figure S8). Additionally, there is no synergistic effect observed in PANC-1 cells with wild-type BRCA2 gene (Figure S9). Thus, this ratio was used in subsequent experimentations with the concentrations of Gem and Ola being 50 ng/mL and 500 ng/mL, respectively. To determine whether the combination of Ola and Gem induced more apoptosis than Gem alone, we assessed Annexin V staining in capan-1 cells after 72 h of treatment. The combination of Ola and Gem led to significantly more apoptosis than either agent alone (Figure 3A,B). For single drug loaded GENP, there was no significant difference on inducing cell apoptosis, compared with the single drug treatment (Figure S10). We also observed a decrease in Bcl-2 and an increase in cleaved caspase-3 accompanied by reduced pro caspase-3 levels in capan-1 cells treated with either Gem+Ola or GENP-Gem-Ola, providing further evidence for enhanced apoptosis in cells treated with both drugs (Figure 3C,D).


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Figure 3. Synergistic inhibitory effects of Gem and Ola in capan-1 cells. (A,B) Apoptosis induced by Gem and Ola in capan-1 cells treated with DMSO, GENP, Gem, Ola, Gem+Ola or GENP-Gem-Ola for 72 h, as assessed by Annexin V-FITC-PI-staining and flow cytometry. (A) Representative images; (B) data from three separate experiments expressed as mean ± S.D. * p < 0.05. (C,D) Western blot analysis of Bcl-2, caspase-3 and cleaved caspase-3 in capan-1 cells after different treatments (as described above) for 72 h. Error bars represent means ± S.D. * p < 0.05, ** p < 0.01.

Combined Gemcitabine and Olaparib Treatment Enhances DNA Damage. Cells with impaired PARP activity require homologous recombination (HR) to resolve SSBs induced by chemotherapy agents and, in its absence, DSBs would accumulate.12 We hypothesized that combined Gem and Ola treatment would enhance the accumulation DNA DSB damage accumulation and thus reduce cell viability. To test this, we used immunofluorescence staining to measure the number of phosphorylated histone H2AX (H2AX) foci formed in the cells in response to various treatments as these foci are characteristic of DSB. Either Gem or Ola alone caused an increase in H2AX nuclear staining (Figure 4A,B), however, the combination of the two drugs led to a higher level of staining, and GENP-Gem-Ola typically showed highest H2AX expression. Western blotting also showed that the level of H2AX was increased in cells treated with GENP-Gem-Ola compared to cells treated with Gem or Ola alone (Figure 4C,D). In addition, there was no significant


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difference on induction of DNA double strands breaks between single drug and single drug loaded GENP (Figure S11).

Figure 4. Combined gemcitabine and olaparib treatment enhances DNA damage accumulation. (A) Immunofluorescence staining for H2AX in capan-1 cells treated with DMSO, GENP, Gem, Ola, Gem+Ola or GENP-Gem-Ola, and representative images are shown. (B) Quantification of the number of H2AX foci positive cells in capan-1 cells treated as indicated. *** p < 0.001. Error bars represent means ± S.D. (C,D) Western blot analysis of H2AX in capan-1 cells treated with DMSO, GENP, Gem, Ola, Gem+Ola or GENP-Gem-Ola. ** p < 0.01. Error bars represent means ± S.D.

Pharmacokinetics of Gemcitabine and Olaparib Co-delivered via GENP. To evaluated biodistribution of the designed nanoparticles, capan-1 tumor-bearing mice were sorted into four groups and injected intravenously with PBS, free Cy5.5, HWNP-Cy5.5, or GENP-Cy5.5 after two weeks inoculation. The tumors and major organs were removed 24 h later and fluorescence signals were recorded using an IVIS Spectrum Imaging System. As shown in Figure S12, the fluorescence in the


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tumors of mice receiving GENP was four fold higher in intensity than that of mice receiving non-targeted HWNP. There was little or no fluorescence in major organs such as the liver, kidney, heart, spleen and lung, suggesting highly specific targeting of GENP-Cy5.5 to tumors. This higher accumulation of GENP-Cy5.5 at the tumor site may be attributed to not only the surface targeting of GENP but also to their nano-size, leading to enhanced accumulation in the tumor via the enhanced vasculature access. Thus, GENP appear to be an ideal candidate nanocarrier for “active” tumor targeting, with the potential to improve antitumor efficacy while reducing the toxicity associated with nonspecific effects on normal cells that accompanies most chemotherapies. To determine the plasma pharmacokinetic profiles of the two drugs with or without their encapsulation within GENP, BALB/c mice were randomly divided into four groups and treated with the free drugs or GENP-Gem-Ola by intravenous injection. After a single injection, blood samples were collected at various time points and the plasma concentration of Gem and Ola were determined using HPLC. As shown in Figure 5A, the circulation time of Gem was much longer when delivered in GENP-Gem-Ola group than when delivered as the free drug (half lives of > 3 h and approximately 1 h for nanoformulation and free drug, respectively). Moreover, the pharmacokinetics of Gem was not influenced by Ola co-administration. Similar results were obtained for Ola (Figure 5B). The half-life of Ola was approximately 1 h in the free drug group, and approximately 4 h in the GENP-Gem-Ola group. In addition, Gem and Ola had the similar pharmacokinetic curves in GENP-Gem-Ola group, suggesting that the two drugs were released simultaneously. These pharmacokinetic results demonstrate that GENP can prolong the circulation time of their cargoes and facilitate coordinated drug release. Achieving an adequate drug concentration within the tumor is a prerequisite for effective cancer therapy. Thus, we assessed the concentrations of Gem and Ola in tumor tissue in mice treated with different drug formulations. Compared with the free drugs, the amount of Gem was twice as higher in the GENP-Gem-Ola group than when free drug was administered (Figure 5C), and it was four fold higher for Ola


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(Figure 5D). Furthermore, in the mice that received GENP-Gem-Ola treatment, the Gem-Ola mass ratio within the tumor was similar to that within the initial GENP-Gem-Ola preparation. To further address this issue, the tumor accumulations of the different drug loaded nanoparticles were evaluated in the orthotopic pancreatic mouse model. As shown in Figure S13, both GENP-Gem and GENP-Gem-Ola groups have significant increased concentration of Gem in tumor tissues compared with Gem group or Gem+Ola group. For Ola, it’s the same that increased concentrations were observed in GENP-Gem and GENP-Gem-Ola groups. Furthermore, single drug loaded GENP showed similar drug accumulation with dual-drug loaded GENP in tumor tissue, which indicated little difference in bio-distribution of these two sized nanoparticles. Taken together, our results indicate that the GENP can not only extend the circulation time of the encapsulated drugs but can also enhance their accumulation at the tumor site in a fixed ratio.

Figure 5. The pharmacokinetics and in vivo tumor accumulations of Gem and Ola in different formulations. (A,B) Plasma concentrations of Gem (A) and Ola (B) at various times after the intravenous administration of either GENP-Gem-Ola or the free drugs (equivalent to 5 mg/kg bodyweight of Gem and 50 mg/kg bodyweight of Ola). The data are presented as the mean ± S.D. (n = 3). (C,D) The concentrations of Gem (C) or Ola (D) in tumor tissue as assessed by HPLC 1 h


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after the administration of the GENP-Gem-Ola or the free drugs. The drug concentrations were normalized to the protein concentration of the tumor. Data are presented as the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01.

Antitumor Effects of GENP-Gem-Ola in Subcutaneous and Orthotopic Tumor Models. To examine whether the encapsulation of the Gem and Ola into GENP could enhance their antitumor effects in vivo, we used BALB/c nude mice bearing capan-1 human pancreatic tumors, implanted subcutaneously approximately 2 weeks prior to treatment. The tumor-bearing mice were randomized into seven groups (n = 5/group). When the tumor volume reached 80 mm3, we began the intravenous administration of the drug preparations (or PBS as a control). The procedure was repeated once every second day, ten times in total. As shown in Figure 6A, the tumors grew progressively and rapidly in PBS-treated mice, those treated with drug free GENP and the free Gem group. The combination of Gem with Ola reduced tumor growth somewhat, but not significantly so compared with the PBS group. Tumors grew even more slowly in the GENP-Gem group but again the reduction in growth was not significant. In the HWNP-Gem-Ola group, tumor grew significantly more slowly than those in saline-treated mice; however, the most dramatic inhibition was observed in the GENP-Gem-Ola group. None of the treatments had any significant effect on the body weight of the mice (Figure 6B). Consistent with the growth curve, the mean weight of the tumors in the GENP-Gem-Ola group was the lowest of all the treatments after the 20-day treatment period (Figure 6C,D). Thus GENP show considerable promise as anti-tumor agents through their highly effective combined delivery of Gem and Ola. Furthermore, GENP mediated drug combination strategy could be used for a wide range of solid tumors as drug combination regimens are typically utilized for improving therapeutic efficacy in clinical cancer treatment. And this stategy provides a ratiometric approach for drug combination to release from nanocarrier with a fixed drug ratio at tumor site with pre-determined genetic defects.


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Figure 6. Enhanced antitumor effects of GENP-Gem-Ola in vivo. (A) Tumor growth curves. Nude mice bearing capan-1 human pancreatic tumors, implanted 2 weeks prior to the commencement of treatment, received intravenous injections of PBS, GENP, Gem, GENP-Gem, Gem+Ola, HWNP-Gem-Ola or GENP-Gem-Ola every other day at a Gem dose of 5 mg/kg and an Ola dose of 50 mg/kg. * p < 0.05, *** p < 0.001. (B) The body weights of the mice were recorded every 2 days throughout the treatment period. (C) Gross morphological appearance of the tumors at the end of the treatment period. (D) Tumors’ weights at the end of the treatment period. n = 5 for each group in (A) and (D). * p < 0.05, ** p < 0.01, *** p < 0.001.

To investigate the mechanisms underlying the suppression of tumor growth by GENP-Gem-Ola, immunohistochemistry was used to assess DSBs (H2AX foci) and apoptosis (cleaved caspase-3 levels and TUNEL) in capan-1 tumor-bearing mice treated with various drug formulations. DSBs appeared as brown nuclear H2AX staining (H2AX row in Figure 7A), while apoptosis was manifest as either brown cytoplasmic (c-Caspase-3 row in Figure 7A) or brown nuclear staining (TUNEL row in Figure 7A). For each parameter, the percentage of positive cells was determined. As shown, accumulated DSBs were observed in tumors of GENP-Gem-Ola group (56%), while the free drugs combination showed less DSBs lesions (28%) (Figure 7B). Tumors from the GENP-Gem-Ola group showed extensive apoptosis (51%


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c-Caspase-3 positive), which was greater than that in tumors from the HWNP-Gem-Ola group (32%). However, only moderate apoptotic signals (23%) were observed in the tumors of mice treated with a combination of the free drugs. In addition, tumors in the GENP-Gem group showed greater apoptosis rate (13%) than those in mice treated with free Gem alone (3%). In the group treated with empty GENP or PBS, only basal levels of apoptosis (about 1%) were seen in the tumors (Figure 7C). Consistent with c-Caspase-3 staining, TUNEL staining showed the similar results (Figure 7D). These results suggest that GENP-Gem-Ola exerts its synergistic inhibitory effects on tumor cells by inducing tumor cells apoptosis.

Figure 7. Immunohistochemical analyses to assess DSBs and apoptosis in capan-1 tumor tissues from mice treated with various drug formulations. (A) Representative images of H&E and immunohistochemistry. DSBs were assessed by staining for H2AX, while apoptosis were examined using c-Caspase-3 and TUNEL. Scale bar = 20 m. (B-D) Quantification of positive staining cells. Data represented as mean ± S.D. n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001.

To further evaluate the antitumor effects of GENP-Gem-Ola in vivo, we injected


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capan-1 cells into the tail of pancreas to simulate the anatomy and pathology of pancreatic tumors. Two weeks after injection, MRI scan of the mice was performed to measure the pancreatic tumors. Mice were then divided into 7 groups (n = 5/group). Various drug formulations were intravenously administered every the other day, eight times in total. At day 16, MRI scan of the mice was performed to assess the tumor burden, then the relative tumor growth rates were analyzed statistically (Figure 8A,C). All mice were then euthanized and the major organs were removed as a whole for ex vivo imaging (Figure 8B). Compared with PBS or GENP group, Gem inhibited the tumor growth to a limited extent. Tumors in GENP-Gem and Gem+Ola group were smaller than those in Gem group. Gem and Ola encapsulated in HWNP showed more suppression of tumor growth than free drug combination. Importantly, the enhancement of antitumor effects in GENP-Gem-Ola group was more significant than HWNP-Gem-Ola. Furthermore, tumor masses were collected and weighed. As shown in Figure 8D,E, the mean tumor weight of GENP-Gem-Ola group was the lowest among all the treatment groups.


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Figure 8. The antitumor effects of GENP-Gem-Ola in the orthotopic model of pancreatic cancer. (A) Representative MRI images of the tumors at the beginning (day 0) and ending (day 16) of the treatment. Capan-1 cells were implanted into the tail of pancreas of BALB/c nude mice. The mice were imaged by MRI scan after 2 weeks of inoculation. Then the mice were divided into 7 groups to receive intravenous injections of PBS, GENP, Gem, GENP-Gem, Gem+Ola, HWNP-Gem-Ola or GENP-Gem-Ola every other day at a Gem dose of 5 mg/kg and an Ola dose of 50 mg/kg. After a two-week treatment period, mice were imaged again with MRI scan. The scale bar represents 1 cm. (B) Representative images of the tumors at day 16. (C) Relative tumor growth rates during the treatment period. n = 5 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., no significance. (D) Gross morphological appearance of the tumors at the end of the treatment period. (E) Tumor weights at the end of the treatment period. n = 5 for each group. * p < 0.05, ** p < 0.01.

Biosafety Evaluation in Vivo. To assess the safety of the various drug formulations following treatment, the heart, liver, spleen, lung and kidney were sectioned, and the histological appearance of the tissues was examined after H&E


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staining. No obvious morphological changes in tissue architecture aforementioned organs were observed in any of the organs in any of the drug formulation treated groups compared to the mice without treatment, indicating that GENP-Gem-Ola did not lead to any gross abnormal effects in the major organs (Figure S14). We also examined biochemical markers of hepatic and renal function in the serum of treated mice (aspartate transaminase, AST; alanine aminotransferase, ALT; blood urea nitrogen, BUN; serum creatinine, Scr) (Figure S15). Treatment with free Gem resulted in an increase in the serum ALT and AST level, indicating liver injury. While, the ALT and AST level in combination of Gem and Ola group was much higher than Gem alone. Importantly, the ALT and AST level in GENP-Gem-Ola group was within the normal range. Thus we conclude that combination of Gem and Ola has aggravated side effects for liver, and GENP can protect the liver toxicity of free drugs.

CONCLUSION We have successfully developed a nanomedicine (GENP-Gem-Ola), with specific tumor targeting and enhanced penetration capacity for simultaneously delivering Gem and Ola to treat BRCA2 mutant pancreatic cancer. The nanomedicine showed excellent stability both in vitro and in vivo and exhibited a strong ability to target cells expressing high levels of EGFR (capan-1 cells) and to selectively accumulate in capan-1 tumor xenografts in mice. We found that the PARP inhibitor Ola was able to enhance cytotoxic effects of Gem and that the combination of Gem and Ola strongly and synergistically inhibited the growth of BRCA2 mutant capan-1 cells in vitro and capan-1 derived tumors in mice. Thus the encapsulation of Gem and Ola in GENP produced a nanomedicine with a long circulation time, strong tumor targeting ability and high antitumor efficacy. It was much more effective than the free drugs and showed almost no side effects on the major organs. This combined drug delivery strategy using nanocarrier offers considerable promise in the clinic where response rate to Gem in unselected PCa patient populations are currently very limited. This


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combination strategy based on synthetic lethality is also applicable for other subgroups of pancreatic cancer patients with other defects in DNA homologous recombination repair mechanism.

EXPERIMENTAL SECTION Preparation and Characterization of Targeting-peptide Based Nanoparticles. The

amphiphilic

peptides

(C18-EEG[YHWYGYTPQNVI]-NH2)

(C18-EEG[HYPYAHPTHPSW]-NH2)

were

synthesized

and

analyzed

and using

high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) and electrospray ionization mass spectrometry by KareBay Biochem (Suzhou, China). According to the quality control reports provided by the manufacturer, the purity was over 90%. To confirm the critical micelle concentration (CMC), C18-EEG-GE11 or C18-EEG-HW12 (0-200 M) was incubated with pyrene (24 g/L) in phosphate buffered saline (PBS, pH 7.4) for 1 h. The fluorescence emission spectrum of each solution was recorded using an excitation wave length of 334 nm with an F-4600 fluorescence spectrophotometer (Hitachi, Japan). The intensity ratio of the first (370-373 nm) to third (381-384 nm) vibronic bands was plotted against the concentration to calculate the CMC. In order to understand the self-assembly of the amphiphilic peptides, 1 mg of C18-EEG-GE11 or C18-EEG-HW12 peptide was dissolved in 10 L of DMSO, followed by dilution into 1 mL of PBS (pH 7.4) under ultrasonication (600 W) for 15 min. The solution was then incubated at room temperature for 1 h. For individual or dual drug loading into the peptide nanoparticles, single drug or dual drugs and peptides were dissolved in DMSO, and a series of parameters, including the amount of each drug, the mass ratios of the dual drugs, and the amount of peptides were investigated to optimize the loading capacity. The compounds were mixed and diluted in PBS (pH 7.4) under ultrasonication (600 W) for 15 min. Then the solutions were transferred into dialysis cartridges with molecular weight cutoff value of 1,000. The cartridges were dialyzed against 10 mL PBS. After incubation for 1 h at room temperature, the solutions were then centrifugated at 6,000


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g for 5 min. The supernatants were collected for further characterization. The morphology of the drug-free nanoparticles and the drug-loaded nanoparticles were characterized by TEM (Tecnai G2 F20 U-TWIN, FEI, USA) using a negative staining method with uranyl acetate. The size and zeta potential of the nanoparticles were evaluated by DLS (Zetasizer Nano ZS90, Malvern, UK). The concentrations of the drugs in the supernatants were measured by HPLC following a standard curve of the free drugs. The encapsulation efficiency (EE) was calculated according to the following formula: EE (%) = (mass of drug encapsulated in nanoparticles / mass of drug added) × 100%. The Drug loading ratio (DLR) was calculated according to the following formula: DLR (%) = (mass of drug encapsulated in nanoparticles / mass of drug added + peptide added) × 100 %. The drug release profiles of GENP-Gem-Ola and HWNP-Gem-Ola were measured in vitro by the dialysis method. Briefly, aliquots of the nanoparticle solutions were injected into dialysis cartridges with a molecular weight cutoff value of 1,000. The cartridges were dialyzed against 10 mL PBS with 10% fetal bovine serum (FBS) and shaken at 37oC. The concentration of Gem and Ola remaining in the dialysis cartridge at different time points was measured by HPLC. The in vitro stabilities of GENP-Gem-Ola and HWNP-Gem-Ola were measured by TEM images after the samples were incubated with 10% FBS/PBS (pH 7.4) at room temperature for 48 h.

Immunofluorescence Analysis of H2AX. Capan-1 cells were seeded into confocal dishes (Nunc, USA), then grown overnight, before been fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature (RT), then blocked with 3% BSA in PBS for 1 h. They were incubated with H2AX antibody (anti-phospho-H2AX(S139), 39117, Active Motif, USA) for 12-16 h at 4°C followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:500) (Molecular Probes) in PBS for 30 min at RT. All fluorescence images were generated using a Carl Zeiss LSM710 scanning laser confocal microscope (Carl Zeiss, Germany).


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Analysis of FITC-labeled Nanoparticle Uptake by Capan-1 Cells. Capan-1 cells were seeded into confocal dish (Nunc, USA). The following day, the cells were washed three times with cold PBS and incubated with FITC-labeled GENP (GENP-FITC) or HWNP (HWNP-FITC) for 15, 30 and 60 min at 37°C. After incubation, the cells were stained with Hoechst 33342 (10 g/mL) for 5 min. Finally, the cells were washed three times with PBS and examined by confocal microscopy (LSM710, Carl Zeiss, Germany). To evaluate the uptake of FITC-labeled nanoparticles quantitatively, 200,000 capan-1 cells were collected in 2 mL centrifuge tubes. After being washed three times with PBS, they were incubated with GENP-FITC or HWNP-FITC for 15, 30 and 60 min at 37°C. Before being analyzed by flow cytometry (BD Accuri C6, BD, USA), the cells were washed three times with PBS and resuspended in PBS. A total of 10, 000 events were collected and analyzed for each sample.

In Vivo Pharmacokinetics and Tumor Drug Concentrations. For in vivo pharmacokinetic studies of Gem and Ola, BALB/c mice were divided into four groups (n = 3). After a single intravenous injection of Gem (5 mg/kg), Ola (50 mg/kg), Gem+Ola or GENP-Gem-Ola (containing equivalent concentrations of Gem and Ola), the mice were anesthetized and blood samples were collected at 0.5, 1, 2, 3, 5, 8, 12 and 24 h post-injection. Blood samples (0.1 mL) were treated with 1.4 mL of isopropanol:water (9:1, v/v) containing 0.075 M hydrochloric acid for 24 h at 4°C to extract Gem and Ola and then centrifuged at 1,000 g for 20 min.30 The concentration of Gem and Ola in the supernatant was determined using HPLC after making a standard curve with relevant free drug. To analyze drug concentrations in tumor tissue, BALB/c mice bearing capan-1 tumor xenografts (two weeks after inoculation) were randomized into four groups. A single dose of Gem (5 mg/kg), Ola (50 mg/kg), Gem+Ola or GENP-Gem-Ola (containing equivalent concentrations of Gem and Ola) was administered via a tail vein. One hour after injection, tumors were harvested and homogenized in RIPA


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buffer (Solarbio, China). The homogenates were centrifuged at 15,000 g for 15 min, and the supernatants were treated using the same procedure employed for blood samples described above. All procedures were performed at room temperature.

Anti-tumor Effects in Vivo. Female BALB/c nude mice (5 weeks old, 18–20 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). All animal studies were approved by the Ethics Committee for Animal Experiments of the Peking University Health Science Center. To establish the tumor xenograft model, freshly harvested capan-1 cells were suspended in 100 L of a 1:1 (v/v) mixture of PBS and Matrigel (BD, USA), and injected subcutaneously into the back of the mice at a single site. To establish the orthotopic tumor model, capan-1 cells were injected into the tail of pancreas through operation. Two weeks after injection, MRI scan of the mice was performed using a 7.0 T Biospec 70/20 USR animal MRI instrument (Bruker, Germany) to measure the pancreatic tumors. The T2_TurboRARE sequence were set as follows: FOV (field of view) = 30 × 30 mm2, MTX (matrix size) = 256 × 256, slice thickness = 1 mm, TR = 2327 ms, TE = 40 ms. Tumor-bearing mice were randomized into different treatment groups (n = 5/group) when the tumor volume reached 80mm3, and the mice were treated with PBS, GENP, Gem, GENP-Gem, Gem+Ola, HWNP-Gem-Ola or GENP-Gem-Ola at a Gem dose of 5 mg/kg and an Ola dose of 50 mg/kg by intravenous injection. The administration was repeated ten timed once every 2 days. The tumor volume and body weight of the mice were recorded every 2 days during the treatment. Tumors were measured using a digital caliper, and tumor volume (V) was calculated as 1/2 (tumor major axis) × (tumor minor axis)2. For orthotopic mouse model, MRI scan of the mice was performed to assess the tumor burden. After a three-week treatment period, all mice were euthanized and the tumors were excised. The tumors were photographed and weighed before being fixed in paraformaldehyde, embedded in paraffin and sectioned for Hematoxylin and Eosin (H&E) staining. To evaluate cancer cell apoptosis, tumor section were stained for cleaved caspase-3 (9664S, CST, USA) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).


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To assess DNA double-strand breaks, the tumor sections were stained for H2AX (39117, Active Motif, USA). To assess the safety of the various treatments, the major organs were sectioned for H&E staining and biochemical markers of hepatic and renal function in the serum of treated mice (aspartate transaminase, AST; alanine aminotransferase, ALT; blood urea nitrogen, BUN; serum creatinine, Scr) were examined.

Statistical Analysis. Statistical analysis was carried out with SPSS 19.0 software (SPSS Inc., Chicago, IL). Significant differences in the data were determined by Student’s t test or ANOVA; p values less than 0.05 were considered significant.

ACKNOWLEDGMENTS This work was supported by the National Key R&D Program of China (2018YFA0208900), Beijing Municipal Science & Technology Commission (Z161100000116035, Z161100000516038), the Excellent Young Scientists Fund (31722021), the National Natural Science Foundation of China (51673051, 21877023, 81572339, 81871954 and 81672353), Beijing Nova Program (Z171100001117010), Beijing Natural Science Foundation (7172164), Youth Innovation Promotion Association CAS (2017056), the Innovation Research Group of the National Natural Science Foundation (11621505), the Key Research Project of Frontier science of the Chinese Academy of Sciences (QYZDJ-SSW-SLH022), the National High Technology Research and Development Program of China (SS2015AA020405).

ASSOCIATED CONTENT Supporting Information Available: Materials and methods (cell culture, cell viability and apoptosis assays, western blot analysis, circulation time and biodistribution of nanoparticles in vivo); Table S1-5 (characterization by DLS,


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encapsulation efficiency); Figure S1-13 (characterization of HWNP, in vitro stability of nanoparticles, confirmation of BRCA2 mutation, EGFR expression, cellular uptake, cytotoxcity of gemcitabine and olaparib, combination index, cell apoptosis, DNA double strands breaks, biodistribution of nanoparticles, the tumor accumulations of drugs, H&E staining of major organs, biochemical indicators related to liver and renal functions. This material is available free of charge via the Internet at https://pubs.acs.org.

Conflict of Interest: The authors declare no competing financial interest.


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Table of Contents Epidermal Growth Factor Receptor Targeting Peptide Nanoparticles Simultaneously Deliver Gemcitabine and Olaparib to Treat Pancreatic Cancer with Breast Cancer 2 (BRCA2) Mutation.


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