Targeted Delivery System Based on Gemcitabine-Loaded Silk Fibroin

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Targeted delivery system based on gemcitabine loaded silk fibroin nanoparticles for lung cancer therapy Fatemeh Mottaghitalab, Melika Kiani, Mehdi Farokhi, Subhas C. Kundu, Rui L. Reis, Mahdi Gholami, Hassan Bardania, Rassoul Dinarvand, Parham Geramifar, Davood Beiki, and Fatemeh Atyabi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10408 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 25, 2017

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ACS Applied Materials & Interfaces

Targeted delivery system based on gemcitabine loaded silk fibroin nanoparticles for lung cancer therapy Fatemeh Mottaghitalab1, Melika Kiani1, Mehdi Farokhi2, Subhas C. Kundu3, Rui L. Reis3, Mahdi Gholami4, Hassan Bardania5, Rassoul Dinarvand1, Parham Geramifar6, Davood Beiki6, Fatemeh Atyabi 1,* 1

Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 2

3

National cell bank of Iran, Pasteur Institute of Iran, Tehran, Iran

3Bs Research Group, Headquarters of the European Institute of Excellence on Tissue

Engineering and Regenerative Medicine, University of Minho, AvePark - 4805-017 Barco, Taipas, Guimaraes, Portugal 4

Faculty of Pharmacy and Pharmaceutical Science Research Center, Tehran university of Medical Sciences, Tehran, Iran

5

Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran 6

Research Center for Nuclear Medicine, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran

•

Correspondence: Prof. Fatemeh Atyabi, Tel/Fax: +982166959059, Email: [email protected] 1 ACS Paragon Plus Environment


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Abstract Here, a targeted delivery system was developed based on silk fibroin nanoparticles (SFNPs) for systemic delivery of gemcitabine (Gem) in order to treat induced lung tumor in mice model. For targeting the tumorigenic lung tissue, SP5-52 peptide was conjugated to Gem- loaded SFNPs. Different methods were used to characterize the structural and physicochemical properties of SFNPs. The prepared NPs showed suitable characteristics in terms of size, zeta potential, morphology, and structural properties. Moreover, targeted Gem-loaded SFNPs showed higher cytotoxicity, cellular uptake, and accumulation in the lung tissue in comparison to non-targeted SFNPs and control groups. Afterwards, a mice model with induced lung tumor was developed by intra-tracheal injection of Lewis lung carcinoma (LL/2) cells into the lungs for assessing the therapeutic efficacy of the prepared drug delivery system. The histopathological assessments and SPECT-CT radiographs showed successful lung tumor induction. Moreover, the obtained results showed higher potential of targeted Gem-loaded SFNPs in treating induced lung tumor compared with the control groups. Higher survival rate, less mortality, and no sign of metastasis were also observed in those animals treated with targeted NPs based on the histological and radiological analysis. This study presented an effective anticancer drug delivery system for specific targeting of induced lung tumor that could be useful in treating malignant lung cancers in future. Key words: Silk fibroin, Gemcitabine, SP5-52 peptide, Lung tumor induction, Lewis lung carcinoma cells.

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1. Introduction Lung cancer has been the main cause of death in men and women over the past decade 1. The number of new cases of lung and bronchus cancer is about 57.3 per 100,000 in a year. There are many risk factors for lung cancer such as smoking, air pollution, family history, exposure to radon gas, and weakened immunity 2. Lung cancers are typically classified into two different categories based on the microscopic appearance of tumor cells including small cell lung cancers (SCLC) and non-small cell lung cancers (NSCLC). NSCLC has higher incidence (~85%) than SCLC and therefore gained more attention for research activities. To date, different strategies have been used to treat NSCLC such as chemotherapy, radiotherapy, monoclonal antibodies, and surgery 3. Generally, NSCLCs have relatively poor sensitivity to chemotherapy and radiotherapy 4,5

due to the suboptimal accumulation of anticancers in different tissues with inevitable side

effects. Therefore, it seems that using local drug delivery systems for specific targeting of tumors would be more applicable and suitable for cancer treatment because it can increase the survival and quality of life, increase the local concentration of drug in the targeted site, and minimize the systemic side effects 6. Recently, various delivery systems such as liposomes, nanoshells, dendrimers, and nanoparticles (NPs) have been developed for effective treatment of NSCLCs

7-9

. Among different

nanostructures used for anticancer delivery, NPs have paid more attention due to their exceptional properties as a drug vehicle such as high surface area to volume ratio, tunable size and surface charge, high drug loading capacity, and ability to target specific tissues10. Applying inhaled NPs was the leading approach for specific delivery of anticancer drugs into cancerous lung tissue

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. However, some features such as size, shape, surface chemistry, and low 3 ACS Paragon Plus Environment


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metabolic activity of lungs limit the application of inhaled NPs for lung cancer therapy. In order to overcome the limitations of conventional inhaled NPs, systemic administration of polymeric NPs with targeted properties have been developed as a potential replacement. Systemic delivery of drug conjugated NPs can improve the bioavailability, stability, half-life, and clearance of drugs from the body. It can also direct the anticancer drugs into the desired tissue by receptormediated endocytosis

13

. Various biomaterials, whether natural or synthetic, can be used for

preparing NPs based drug delivery systems 14-16. In recent years, natural polymer based NPs have been more interested in biomedical fields due to their suitable biocompatibility, biodegradability, less immunogenicity, and the ability to well-tolerate with biological systems

17-20

. In the present

study, we used silk fibroin NPs (SFNPs) for systemic administration of gemcitabine (Gemzar; Gem) to lung tumor. SF possesses many unique properties such as suitable biocompatibility, tunable biodegradability, low immunogenic response, and high cellular uptake

21-24

.

Gemcitabine, as an FDA approved anticancer drug, has high potential in suppressing NSCLCs 25. For targeting the prepared formulation to the cancerous lung tissue, SP5-52 peptide was also conjugated to SFNPs. SP5-52 is a highly stable peptide with small size and improved therapeutic efficacy among other targeting agents such as aptamers, siRNA, antibodies, and etc.

26

. SP5-52

peptide is able to direct anticancer drug carriers to non-small-cell lung cancer (NSCLC) cells which can 5.7-fold increase the accumulation of anticancers in tumorigenic lung tissue in comparison to non-targeted carriers

27

. Lee et al. reported that SP5-52 peptide-linked liposome

loaded with doxorubicin had high potential in targeting human lung tumors

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. Therefore, we

hypothesized that using SP5-52 peptide conjugated SFNPs would be an effective systems for specific delivery of Gem into cancerous lung tissue. Applying this delivery system not only can improve the therapeutic efficacy of Gem but also can reduce its various side effects on normal 4 ACS Paragon Plus Environment

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tissues. A novel method is also presented in this study for inducing lung tumor in mice model by intra-tracheal injection of Lewis lung carcinoma cells (LL/2). Based on our knowledge, some methods were previously used for inducing lung tumor in animal models such as intra-bronchial injection of cancerous cells and thoracotomy. However, intra-tracheal injection of LL/2 cell into lung was more feasible and effective in preserving the viability of cell during infusion that lasted only 3-5 minutes. Moreover, it is suggested that intra-tracheal injection of LL/2 cells has more efficiency in inducing lung tumor in comparison to other methods. Here, we seek to characterize physical, chemical, and biological properties of targeted Gemloaded SFNPs in vitro. Afterwards, the efficacy of tumor induction in mice model using SPECTCT scan and histopathological analysis was evaluated. Finally, the potential of designed delivery system in treating induced lung carcinoma in mice model was assessed using SPECT-CT scan and histopathological assessment. The rate of survival and the histological evaluation confirming the level of metastasis were also addressed. 2. Experimental procedures 2.1 Materials Sodium carbonate, lithium bromide (LiBr), dialysis tube (cut off 12KDa), formic acid, phosphate buffered saline (PBS), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), ketamine, Xylazine, isopropanol, and anhydrous dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (USA). Dulbecco Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (USA). Moreover, LL/2 cell line (Lewis lung



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carcinoma) and BEAS-2B cells (Lung/bronchus fibroblasts) were obtained from National Cell Bank of Iran (NCBI). 2.2 Methods 2.2.1 Preparation and characterization of silk fibroin nanoparticle Prior to SFNPs preparation, silk fibroin was extracted from Bombyx mori cocoons according to the previously reported work

19

. Briefly, Bombyx mori cocoons were degummed with 0.02%

(w/v) Na2CO3 solution for 60 min and washed with distilled water. The degummed silk was then dissolved in 9.3 M LiBr for 4h at 60 ºC and dialyzed against distilled water for 72h at room temperature. Consequently, SFNPs were prepared by dissolving 10 mL anhydrous DMSO in 10 mL of SF solution (2% w/v) at room temperature with constant stirring at a very low rpm. The solution were centrifuged at 23500×g for 10 min to participate SFNPs aggregates which were then washed with centrifugation at 13400×g for 10 min in deionized water to remove the residual DMSO. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) were performed in order to evaluate the size, surface charge, and morphology of SFNPs. 2.2.2 Preparation of Gemcitabine-loaded silk fibroin nanoparticles In order to prepare Gem-loaded SFNPs, 2.5 mg of Gem and 25 mg SFNPs were added to 1mL and 10 mL DMSO, respectively. The mixture was then sonicated for 10 min under 40 Hz frequencies. Afterwards, the solution was dialyzed against water overnight at room temperature using 12KD dialysis tube. The amount of loaded Gem on SFNPs was further determined at 268 nm using UV spectroscopy. The drug loading capacity and encapsulation efficiency were measured according to the below equations: 6 ACS Paragon Plus Environment

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Drug loading (%) =

Encapsulation efficiency (%) =

× 100

Equation (1) × 100

Equation (2)

The size and zeta potential of drug loaded SFNPs were evaluated using DLS. The morphology of Gem-loaded SFNPs and the conjugation between Gem and SFNPs were also assessed by SEM and Fourier transform infrared (FTIR). 2.3.3 In vitro release study The release profile of Gem from SFNPs was assessed in phosphate buffer saline (PBS) at pH 5 and 7.4. This study was performed by dissolving 125 mg Gem-loaded SFNPs in 1 mL PBS solution. The samples were kept in a shaker incubator (Heidolph, unimax 1010) at 37 ºC and 100 rpm. At each time intervals, 500 µL of samples were withdrawn and replaced with the same amount of fresh buffer and finally analyzed by UV spectroscopy at 268 nm. The sink condition was maintained during the study. 2.2.4 Synthesis and characterization of SP5-52 peptide The SP5-52 peptide sequence (SVSVGMKPSPRP) was manually synthesized according to standard 9-fluoromethoxycarbonyl (Fmoc) chemistry

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. SP5-52 peptide was synthesized by

solid-phase peptide synthesis on Rink Amide MBHA resin. HATU and DIEA were used as coupling reagents and Kaiser ninhydrin test used for monitoring coupling efficiencies. The final synthesized peptide was cleaved from the resin by treatment with cleavage cocktaile (TFA/H2O/TIPS = 95/2.5/2.5) for 2h and resin removed by filtration. The crude peptide was precipitated in cold diethyl ether and lyophilized for 24h. The molecular weight of the peptide was confirmed by LC-MS spectrometry (Agilent Technology Series). 7 ACS Paragon Plus Environment


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2.2.5 Preparation of targeted Gem-loaded SFNPs SP5-52 peptide was covalently attached to SFNPs using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Briefly, 10 mg of SFNPs were dissolved in DMSO and 0.4 mg of EDC (final concentration 2 mM) and 0.6 mg of NHS was added directly to the solution, resulted in a 10-fold molar excess of EDC to SFNPs. It took 15 min to complete the reaction between the components at room temperature. The SP5-52 was then mixed with the solution containing activated SFNPs and the reaction allowed to proceed for 2h at room temperature. DMSO and excess EDC were removed by dialysis against water for 48h. To confirm the conjugation, Raman spectroscopy (Almega Thermo Nicolet Dispersive Raman Spectrometer; Germany) was performed. Moreover, DLS and SEM were utilized to examine the size, zeta potential, and morphology of targeted SFNPs. FTIR was also performed in order to assess the structural changes of SFNPs after SP5-52 peptide conjugation. 2.2.6 In vitro toxicity Prior to cell studies, the 2×104 Lewis lung carcinoma cells (LL/2 cell line) and BEAS-2B cells were cultured in DMEM with 10% FBS and incubated at 37 ºC with 5% CO2. After 24h, the in vitro cytotoxicity of prepared SFNPs was measured using MTT assay. For these studies, four groups including free Gem, SFNPs, Gem-loaded SFNPs, and targeted Gem-loaded SFNPs were considered. Tissue culture polystyrene (TCPS) was also used as negative control group. 2.2.7 Cellular uptake The SFNPs were first labeled with fluorescein isothiocyanate (FITC) before studying the cellular uptake. For this, FITC (100 mg) was dissolved in 10 mL DMSO and added to SFNPs suspension 8 ACS Paragon Plus Environment

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(1mg/mL). The prepared solution was remained in dark for 10h at room temperature. Afterwards, NaOH (0.5 mM) was used to precipitate the FITC-labeled SFNPs followed by dialysis (12KDa dialysis tube) against water for 3 days. The cells were then allowed to adhere to a glass cover slip in 6-well plate for 24h. The medium was removed and the cells were incubated with 100 µg/mL of each sample for 4h. Afterward, the samples were washed 3 times with PBS and then incubated with DAPI for 5 min in order to stain the nuclei and rewashed 3 times with PBS. The cells were fixed with 1% formaldehyde for 10 min at 4°C and the cellular uptake was assessed using confocal fluorescence scanning microscope (CFSM; Nikon, Switzerland). In order to better understand the mechanism of cellular uptake of the prepared formulations, the BEAS-2B cells (normal epithelial lung cells) were used as control group. Flow-cytometric analysis was then implemented to quantitatively compare the uptake of targeted and non-targeted SFNPs in both cells. 2.2.8 Lung tumor induction For lung tumor induction, 20 Balb/C mice weighting about 15±2g were used. The immunity system of animals was suppressed using 25 mg/mL cyclosporine three days before cell injection. For intra-tracheal injection of cells, the animals were firstly anesthetized using standard amount of Ketamine and Xylazine and then the frontal segment of neck was exposed. Afterwards, 3×105 of LL/2 cells was injected into the lungs using 16G polyethylene catheter. The incisure was then sutured and the animals were maintained under sterilized condition (Figure 1).



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Figure 1. (A-D) Surgical process of intra-tracheal implantation of LL/2 carcinoma cells for inducing lung tumor in Balb/C mice. The animals received antibiotic for five days after surgery. After 10 days, the efficacy of tumor induction and the size of tumors were evaluated using histopathological analysis and SPECT-CT scan. Table 1 summarizes the number of injected cells, number of injected animals, number of animals with tumor, and the rate of animal mortality during the cellular injection. Table 1. The number of injected cells and the rate of mortality during cell implantation.

Group Group 1 Group 2 Group 3 Group 4

Number of implanted cells 3×105 3×105 3×105 3×105

Number of injected animals 10 10 10 10

Number of animals with tumor 8 9 8 8


Mortality rate 20% 10% 20% 20%

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2.2.9 In vivo whole body tissue distribution In vivo whole body imaging of animals was used to assess the accumulation of targeted and nontargeted SFNPs in different tissues of the animal model. Prior to the study, the prepared formulations were labeled with FTIC according to the protocol mentioned in section 2.2.7. Balb/C mice weighting about 15±2g were used for this study which were fasted overnight but had free access to water. The animals were anesthetized using standard amount of Ketamine and Xylazine and then 100 µL of FITC-labeled formulations were injected intravenously (I.V.) from the tail vein of the animals. The whole body animal imaging (Kodak; In vivo imaging system, FPro) was performed 1 h after injection. 2.2.10 Evaluating the efficacy of the prepared nanoparticles in treating induced lung tumor The main goal of this study was to evaluate the efficacy of Gem- loaded SFNPs, whether targeted or non-targeted, for induced lung cancer treatment. For this purpose, the animals were assigned into four different treatment groups including: 1) PBS, 2) Gem, 3) Gem- loaded SFNPs, and 4) targeted Gem- loaded SFNPs. The therapeutic efficacy of the prepared SFNPs was evaluated after 1 month post-surgery using SPECT-CT scan. Moreover, some of the animals from each group were randomly chosen and the lung was exposed after scarification for histological analysis. Moreover, the weight loss, the amount of water and food consumption, and the rate of mortality were measured during 3 months. 2.2.11 Statistical analysis The quantitative data were expressed as means ± standard deviation using one-way ANOVA with SPSS 16.0 (SPSS, USA). P

Targeted Delivery System Based on Gemcitabine-Loaded Silk Fibroin

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