|Year : 2014 | Volume
| Issue : 1 | Page : 15-20
The investigation of epsilon toxin effects on different cancerous cell lines and its synergism effect with methotrexate
Azin Gholami Shekarsaraei1, Sadegh Hasannia2, Nazanin Pirooznia1, Fariba Ataiee3
1 Department of Biology, Faculty of Sciences, The University of Guilan, Rasht, Iran
2 Department of Biochemistry, School of Biological Sciences, Tarbiat Modares University; National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
3 National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
|Date of Web Publication||23-Apr-2014|
Department of Biochemistry, School of Biological Sciences, Tarbiat Modares University, Jalal Ale Ahmad Highway, Tehran
Source of Support: None, Conflict of Interest: None
Background: The overall goal of this study is to use a bacterial toxin as drug delivery agents for chemotherapy drugs and overcome the development of resistance to these medicines. COR-L105 and MDA-MB 231 which are epithelial-like were used in this study. Cytotoxicity assays were performed by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as metabolic indicator. The toxin was essential to kill 50% (CT50) and IC 50 value (inhibition growth value) for methotrexate were determined as optical density at 540 nm. Epsilon toxin-loaded PLGA nanoparticles were prepared using non-aqueous technique. Surface morphology, in vitro drug release, and encapsulation efficiency of the nanoparticles was determined.
Results: Results confirmed that using non-toxic concentration of epsilon toxin, resistance to cancerous cell decreased significantly, which could be an important result in cancer therapy. The synergistic effect of MTX and epsilon toxin showed that bio toxins can be used as supplement with chemical drugs and increase the effect of chemotherapy. The results illustrated that application of PLGA as drug delivery system due to its controlled release properties was beneficial.
Conclusion: These finding proposed that due to the ease of local accessibility of lung tumors with aerosol drug delivery, biotoxins can directly be used with chemotherapy drugs in aerosol form.
Keywords: Epsilon toxin, methotrexate, nanoparticle, Poly Lactic-co-Glycolic Acid, synergistic
|How to cite this article:|
Shekarsaraei AG, Hasannia S, Pirooznia N, Ataiee F. The investigation of epsilon toxin effects on different cancerous cell lines and its synergism effect with methotrexate. J Can Res Ther 2014;10:15-20
|How to cite this URL:|
Shekarsaraei AG, Hasannia S, Pirooznia N, Ataiee F. The investigation of epsilon toxin effects on different cancerous cell lines and its synergism effect with methotrexate. J Can Res Ther [serial online] 2014 [cited 2020 Feb 26];10:15-20. Available from: http://www.cancerjournal.net/text.asp?2014/10/1/15/131338
| > Introduction|| |
Cancer is caused by uncontrolled growth and spread of abnormal cells. It is estimated that cancer leads to more than half million death annually. , Appropriate treatments for cancer involve surgery, radiotherapy, and chemotherapy.  Chemotherapy medications work by killing rapidly dividing cells. Since cancer cells divide more frequently than most cells, they are particularly susceptible to these drugs. In recent decades, methotrexate is used as an effective chemotherapeutic agent in a variety of cancers specially breast, lung, head and neck, and sarcomas. Methotrexate is an anti-metabolic and folic acid antagonist, which inhibits the enzyme dihydrofolate reductase (DHFR) and stops DNA synthesis. Unfortunately, resistance to this agent is common, representing a major obstacle to successful treatment. The main factors which restrict the efficiency of treatment are development of drug resistance, non-specific targeting and toxicity. ,, Hence, other techniques are being expanded to target and overcome tumor.  One of the potential therapeutic strategies is the application of bacterial pore-forming peptides for destruction of cancer cells. Cytotoxicity of these peptides is performed by forming cytolytic pores in the membrane or by increasing penetration of chemotherapeutic agents to cancer cells.  Epsilon toxin type B and D belongs to cytolytic pore-forming proteins, which are produced by Clostridium perfringens. It is synthesis as an inactive protoxin (32.9 kDa) and is converted to a highly active mature protein through proteolytic removal of 10-13 amino-terminal and 22-23 C-terminal amino acids with proteases such as trypsin, chymotrypsin, and λ-protease produced by C. perfringens types B and D. , Epsilon toxin can lead to enterotoxemia in a variety of domestic animals, but it is unclear if epsilon toxin has threatening effects on human or not. , However, an anti-ε-toxin antibody is produced to protect against this toxin. , Epsilon toxin has been spread by activation into blood-stream, affecting the lungs, kidneys, and the brain.  Active toxin binds predominantly to detergent-resistance membranes (DRMs) or lipid rafts. Previous studies suggest that accumulation of epsilon toxin receptor in DRMs induces its heptamerization (forms a heptameric complex in cell membrane). , Two susceptible cell line such as Madin Darby canine kidney (MDCK) and human renal leiomyoblastoma G-402 cells have specific membrane receptor for epsilon toxin. , β-barrel pore being formed by epsilon toxin leads to K + efflux from cells and influx of Na + and Cl - ions into the cells. ,,
Besides, the main challenges in drug targeting are poor solubility, short half-life, and severe side-effects. Pharmaceutical use of peptides and proteins has some limitation due to their rapid degradation, low half-life, and emission from biological barrier; therefore, the use of nanoparticles (NPs) as drug carriers for in vitro and in vivo sustained drug release is recommended. Pharmaceutical carriers concentrate on target site, and then drug can be released through enzymatic activity and temperature or pH change. , A continuous delivery and increase in the bioavailability of insoluble drugs are the main advantage of PLGA as drug delivery system and provide maximum efficiency in minimum drug dose.  Additionally, the administration of NPs helps to increase stability of drug/protein.  Moreover, It has been confirmed by US FDA for drug delivery.  PLGA is one of the most common polymers used in drug delivery purposes, which is biocompatible and biodegradable. The degradation rate of PLGA depends on monomer ratio (lactide/glycolide), molecular weight, degree of crystallinity, and the transition glass temperature (Tg) of the polymer used.  Hydrolytic activity is enhanced when the NP is prepared by 50:50 ratio of lactic and glycolic acids. , Minimal systemic toxicity of PLGA is due to the rapid metabolism of degradation products through the tricarboxylic acid cycle. , Several methods have been administrated for the preparation of biodegradable PLGA, but the main techniques are used including phase separation (coacervation), spray drying, and solvent evaporation. water-in-oil-in-water (W/O/W) or oil-in-oil (O/O) emulsions which is a solvent evaporation method can be applied for encapsulating hydrophilic drugs with high efficiency.  There are several methods for the release of drug molecules from drug delivery system based on PLGA such as transport through water-filled pores, transport through the polymer, and due to solution of the encapsulating polymer.  PLGA copolymer is converted to its oligomers and finally monomers by cleavage of its backbone ester linkages. 
The purpose of this study is to investigate the effect of epsilon toxin alone and with MTX in the reduction or complete destruction of cancerous cells. On the other hand, the synergistic effect of epsilon toxin and MTX on cancer cells was also examined. Epsilon toxin-loaded nanoparticle was prepared by non-aqueous technique using 50:50 ratio of lactic acid/glycolic acid. Particle size, surface morphology, entrapment efficiency, and in vitro release of epsilon toxin-PLGA-NPs were evaluated.
| > Materials and Methods|| |
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640, α-MEM, fetal bovine serum (FBS), and penicillin/streptomycin were procurement from Gibco BRL (now part of Invitrogen Corporation, Carlsbad, CA, USA), and DMSO was bought from Merck (Germany). Poly (D, L-lactide-co-glycolide) (PLGA) with a copolymer ratio of lactide: glycolide 50:50 (Mw 40,000-75,000) was purchased from Sigma Aldrich (USA). Span80, n-hexane, viscous Paraffin, and Acetonitrile were purchased from Merck (Germany). Chicken anti-rabbit IgG FITC-labeled was obtained from Abnova.
Activation of purified toxin was done using 10 mg/ml trypsin (GIBCO) and incubated for 1h in 37˚ C. The cells lines that have been used in these experiments were COR-L105, MDA-MB 231 lung and breast epithelial-like cells, which were obtained from Pasteur Institute, Iran. Both of them were cultured in RPMI 1640 media containing 2 mM L-glutamine, NaHCO 3 and 100 units/ml penicillin, 100 mg/ml streptomycin and supplemented with 10% FBS (GIBCO) and incubated at 37°C in 5%/CO 2 humidified incubator.
The Methoterate efficacy and activity of trypsin-activated epsilon toxin (achieved from Razi Institute, Iran), epsilon toxin-loaded PLGA were assessed using COR-L105, MDA-MB 231 by MTT assay. Using 3 × 10 4 cells per well in 96-well plates, serial dilution of epsilon toxin, epsilon toxin-loaded PLGA, and MTX were prepared in the α-MEM media with 1% FBS. Cytotoxicity assays were performed using MTT assay as metabolic indicator. The toxin doses required to kill 50% (CT50) and IC 50 value (inhibition growth value) for methotrexate were determined by optical densities at 540 nm.
Identification and localization of antigens in cells was done using Immunocytochemistry. In this method, the position and presence of the target protein in cell surface can be observed using specific labeled antibodies (primary or secondary). Control and treated cells were treated with epsilon toxin and were fixed by 4% par formaldehyde for 20 min and then washed a couple of times with PBS. After washing, blocker solution was added for 3h at room temperature. The primary antibodies were poured on cells (1/2000 in 300 μL PBS solution), and then the cells were placed in 4°C for overnight to bind specifically. Washing step was repeated once as above and incubated with secondary FITC-labeled antibody with DAPI stain for 2h. After washing, cover slips were mounted on slides and analyzed using a fluorescence microscope.
PLGA nanoparticles containing epsilon toxin were prepared by non-aqueous emulsion technique. In this method, 25 mg of PLGA (50:50) was mixed with 5 ml of acetonitrile and allowed to stir up to 30 min. An activated form of epsilon toxin (2 mg) was dissolved in double distilled water and was added drop-wise to the abovementioned solution. The above mixture was added by consecutive drop-wise addition to a stirred solution of 18 ml viscose paraffin containing %1 v/v Span80. Stirring was continued for 2h to ensure the solvent evaporation and nanoparticle hardening. Nanoparticles were collected by centrifuge at 18000 rpm for 30 min, washed twice with n-hexane to remove mineral oil, freeze-dried (LaboGene ScanVac CoolSafe freeze dryer) and stored at −20° C.
Morphological investigation of nanoparticles was carried out by using scanning electron microscopy (TESCAN Vega LMU, USA). One drop of the nanoparticles suspension was deposited on an aluminum stub and placed in a desiccator to obtain a uniform layer of nanoparticles. Samples were coated with gold using EMITECH K450X sputter-coater (England).
Determination of loading efficiency was carried out by dissolving 5 mg of prepared nanoparticle in 0.5 ml of NaOH 1 M. The following solution was incubated overnight at 37°C and was neutralized by adding 0.5 ml of HCl 1M. Supernatant obtained by centrifugation (5 min at 13000 rpm) was evaluated for epsilon toxin concentration. The concentration of epsilon toxin present in each sample was calculated by measuring the absorbance on a spectrophotometer at 595 nm.
In vitro release of epsilon toxin from PLGA was conducted in phosphate buffer saline pH 7.4 at 37°C. 5 mg loaded PLGA nanoparticles were placed in test tube containing PBS. Tubes were incubated in a shaker water bath (Memmert, Germany) at 37°C up to 15 days. At pre-determined time intervals, 1 ml of sample was removed and replaced with 1 ml of fresh release medium.
| > Results|| |
Epsilon toxin activation
The activation of epsilon toxin by 10 mg/ml trypsin was examined using SDS-PAGE, and the result was shown in [Figure 1].
|Figure 1: Activation of epsilon toxin was confirmed by SDS-page. 1) Protoxin, 2) active toxin|
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Study of cytotoxicity
The single and combination cytotoxicity of MTX, trypsin-activated epsilon toxin were achieved using COR-L105, MDA-MB 231 lung and breast epithelial-like cells by MTT assay. To define CT 50 and LC 50 of epsilon toxin and MTX, cells were treated by various dilutions. The CT 50 and LC 50 that were measured by optical densities at 540 nm shown that MTX and epsilon toxin can be cytotoxic at concentrations 800 and 900 μM and 24 and 28 μg/ml for MDA-MB 231 and COR-L105, respectively [Figure 2].
|Figure 2: Cytotoxicity of MTX and epsilon toxin and both of them was assessed using MTT assay|
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The binding of epsilon toxin to cell membrane receptors was investigated using Immunocytochemistry method. The presence of epsilon toxin on cells (COR-L105, MDA-MB231) surfaces was confirmed by FITC-labeled antibody [Figure 3].
|Figure 3: Position and presence of the epsilon toxin on cell membrane was observed using Immunocytochemistry technique. In (a, c) control cells (COR-L105, MDA-MB231) and in (b, d) cells (COR-L105, MDA-MB231) treated with epsilon toxin, respectively|
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Surface morphology and size of epsilon toxin-loaded-PLGA nanoparticles were determined using SEM. The surface of the sphere-shaped nanoparticles appeared to be smooth without pores. Mean particle size varied between 150 and 250 nm as confirmed by SEM [Figure 4].
|Figure 4: Morphology of PLGA spheres revealed by scanning electron microscopy|
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The entrapment efficiency of epsilon toxin-loaded-PLGA was revealed by dissolving PLGA NPs containing epsilon toxin into NaOH. Entrapment efficiency and protein concentration were obtained by spectrophotometer as shown in [Table 1].
The entrapment efficiency was determined by the following equation:
Release profiles were recognized by release medium, phosphate buffer saline at pH 7.4. The release study showed that initial burst phase occurred in 8 hours of initial release, and this finding indicates that the sustain release from NPs continued up to 15 days. The concentration of the released protein was evaluated using spectrophotometer [Figure 5].
|Figure 5: In vitro release profiles of epsilon toxin from PLGA nanoparticles with 50:50 ratio synthesized by non-aqueous emulsion technique|
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The cell cytotoxicity was assessed by applying different concentration of PLGA NPs on lung epithelial-like cells COR-L105 and MDA-MB 231 breast epithelial-like cells. The results revealed that the viability of cells treated with serial concentrations of free PLGA NPs remains unchanged, but epsilon toxin-loaded PLGA nanoparticles was cytotoxic for both cell lines, and the number of viable cells decreased significantly [Figure 6].
|Figure 6: Estimated cytotoxicity of epsilon toxin-loaded PLGA on cells (1) COR-L105, 2) MDA-MB231 was evaluated by MTT assay|
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| > Discussion|| |
Cancer is the second most common cause of death after heart disease and is the uncontrolled growth of cells. ,, Surgery, radiotherapy, and chemotherapy are conventional treatment of cancer.  During chemotherapy, cancer cells can acquire resistance. Methotrexate is a classical folate antagonist, which has a wide application in the treatment of various types of solid tumors, either alone or in combination with other chemotherapeutic agents. , A major problem in cancer therapy is the emergence of tumor drug resistance with MTX. , Hence, several other strategies have been developed to overcome this problem. Bacterial toxins have appeared as an encouraging cancer treatment strategy.  Pore-forming proteins have been suggested as therapeutic agent for treatment of diseases specially cancer. Epsilon toxin, which is encoded by etx gene placed on large plasmid, belongs to Clostridium perfringens type B and D. In addition, it is secreted as an inactive precursor and changes to an active protein by serine proteases such as trypsin, chymotrypsin, and λ-protease produced by C. perfringens and responsible for enterotoxaemia in domestic animals. , The Madin-Darby canine kidney (MDCK) cell line and human renal leiomyoblastoma (G-402) which have specific receptor binding site on detergent-resistant membrane domains (DRMs) are susceptible to epsilon toxin. However, the toxin does not enter the cytosol and remains connected with the cell membrane by forming a large complex. ,,, C. perfringens can be discovered in the intestines of domestic animals. A small amount of epsilon toxin in the intestine has not any clinical features until bacterial balance is altered; in this case, bacteria proliferate rapidly and produced large amounts of this toxin. Epsilon toxin increases intestinal mucosal permeability and facilitating its absorption into the circulation, then it has been spread into blood-stream, affecting the lungs, kidneys, and the brain. , Epsilon toxin has three-dimensional structural similarity to aerolysin, which belongs to family of toxins known as β-pore-forming toxins. In contrast to aerolysin, epsilon toxin causes cell necrosis instead of apoptosis. Moreover, epsilon-toxin leads to quick depletion of ATP, motivates the AMP-activated protein kinase (AMPK), and influences cell signal pathways through mitochondrial membrane permeabilization.  As drug candidates, peptides and proteins have achieved great attention in recent years and played a key role in biological process. The main limitations of protein drugs application in the treatment of cancer are low stability of proteins in the circulation and poor absorption by targeted cells. , An appropriate drug administration does not generally provide rate-controlled release or target specificity. Furthermore, carrier technology suggests an intelligent approach for drug delivery by joining the drug to a carrier particle such as microspheres, nanoparticles, liposomes, etc., and the adjustment of release and absorption trait of the drug is carried out by carrier particle.  PLGA is approved by the FDA because of its biocompatible, biodegradable, and stable characteristics.  The physical and chemical behavior of PLGA such as molecular weight, glass transition temperature, and copolymer ratios are critical to the biodegradation trait of the polymers. 
| > Conclusions|| |
In this project, results show that combination therapy of MTX with other drugs that could modulate the expression of genes included in MTX resistance would be an important and effective strategy to prevent the development of resistance. 
| > Acknowledgments|| |
The author thanks the support of National Institute of Genetic Engineering and Biotechnology and pharmaceutical company Osveh. Also, author thanks Ms. Maryam Shahali for her assessment and guide. This work was financially supported by University of Guilan.
| > References|| |
|1.||Ehdaie B. Application of nanotechnology in cancer research: Review of progress in the National Cancer Institute′s Alliance for Nanotechnology. Int J Biol Sci 2007,3:108-10. |
|2.||Zhang J, Lan CQ, Post M, Simard B, Deslandes Y, Hsieh TH. Design of nanoparticles as drug carriers for cancer therapy. Cancer Genomics Proteomics 2006;3:147-57. |
|3.||Patyar S, Joshi R, Byrav DS, Prakash A, Medhi B, Das BK. Bacteria in cancer therapy: A novel experimental strategy. J Biomed Sci 2010;17:21. |
|4.||Singh UV, Udupa N. In vitro characterization of methotrexate loaded poly (lactic-co-glycolic) acid microspheres and antitumor efficacy in Sarcoma-180 mice bearing tumor. Pharm Acta Helv 1997;72:165-73. |
|5.||Bertino JR, Goker E, Gorlick R, Li WW, Banerjee D. Resistance mechanisms to methotrexate in tumors. Oncologist 1996;1:223-6. |
|6.||Lu JM, Wang X, Marin-Muller C, Wang H, Lin PH, Yao Q, et al. Current advances in research and clinical applications of PLGA-based nanotechnology. Expert Rev Mol Diagn 2009;9:325-41. |
|7.||Majd S, Yusko EC, Billeh YN, Macrae MX, Yang J, Mayer M. Applications of biological pores in nanomedicine, sensing, and nanoelectronics. Curr Opin Biotechnol 2010;21:439-76. |
|8.||Bokori-Brown M, Savva CG, Fernandes da Costa SP, Naylor CE, Basak AK, Titball RW. Molecular basis of toxicity of Clostridium perfringens epsilon toxin. FEBS J 2011;278:4589-601. |
|9.||Miyata S, Matsushita O, Minami J, Katayama S, Shimamoto S, Okabe A. Cleavage of a C-terminal peptide is essential for heptamerization of Clostridium perfringens epsilon-toxin in the synaptosomal membrane. J Biol Chem 2001;276:13778-83. |
|10.||Cavalcanti MT, Porto T, Porto AL, Brandi IV, Lima Filho JL, Pessoa Junior A. Large scale purification of Clostridium perfringens toxins: A review. Rev Bras Ciênc Farm 2004;40:151-64. |
|11.||McClain MS, Cover TL. Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin. Infect Immun 2007;75:1785-93. |
|12.||Souza AM, Reis JK, Assis RA, Horta CC, Siqueira FF, Facchin S, et al. Molecular cloning and expression of epsilon toxin from Clostridium perfringens type D and tests of animal immunization. Genet Mol Res 2010;9:266-76. |
|13.||Lonchamp E, Dupont JL, Wioland L, Courjaret R, Mbebi-Liegeois C, Jover E, et al. Clostridium perfringens epsilon toxin targets granule cells in the mouse cerebellum and stimulates glutamate release. PLoS One 5 (9): e13046. doi: 10.1371/journal.pone. 0013046. |
|14.||Chassin C, Bens M, de Barry J, Courjaret R, Bossu JL, Cluzeaud F, et al. Pore-forming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells. Am J Physiol Renal Physiol 2007;293:F927-37. |
|15.||Mathur DD, Deshmukh S, Kaushik H, Garg LC. Functional and structural characterization of soluble recombinant epsilon toxin of Clostridium perfringens D, causative agent of enterotoxaemia. Appl Microbiol Biotechnol 2010;88:877-84. |
|16.||Folkman J, Long DM. The use of silicone rubber as a carrier for prolonged drug therapy. J Surg Res 1964;4:139-42. |
|17.||Dhanaraju MD, Rajkannan R, Selvaraj D, Jayakumar R, Vamsadhara C. Biodegradation and biocompatibility of contraceptive-steroid-loaded poly (DL-lactide-co-glycolide) injectable microspheres: In vitro and in vivo study. Contraception 2006;74:148-56. |
|18.||Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE. Biodegradable polymeric nanoparticles as drug delivery devices. J Control Release 2001;70:1-20. |
|19.||Makadia HK, Siegel SJ. Poly Lactic-co-Glycolic Acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers (Basel) 2011;3:1377-97. |
|20.||Mansour HM, Rhee YS, Wu X. Nanomedicine in pulmonary delivery. Int J Nanomedicine 2009;4:299-319. |
|21.||Shive MS, Anderson JM. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev 1997;28:5-24. |
|22.||Mohanraj V, Chen C. Nanoparticles: A Review. Trop J Pharm Res 2006;5:561-73. |
|23.||Fredenberg S, Wahlgren M, Reslow M, Axelsson A. The mechanisms of drug release in poly (lactic-co-glycolic acid)-based drug delivery systems-a review. Int J Pharm 2011;415:34-52. |
|24.||Asai S, Miyachi H, Kobayashi H, Takemura Y, Ando Y. Large diversity in transport-mediated methotrexate resistance in human leukemia cell line CCRF-CEM established in a high concentration of leucovorin. Cancer Sci 2003;94:210-4. |
|25.||Mencia N, Selga E, Rico I, de Almagro MC, Villalobos X, Ramirez S, et al. Overexpression of S100A4 in human cancer cell lines resistant to methotrexate. BMC Cancer 2010;10:250. |
|26.||Thibodeau PA, Bissonnette N, Bedard SK, Hunting D, Paquette B. Induction by estrogens of methotrexate resistance in MCF-7 breast cancer cells. Carcinogenesis 1998;19:1545-52. |
|27.||Soler-Jover A, Blasi J, Gomez de Aranda I, Navarro P, Gibert M, Popoff MR, et al. Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo. J Histochem Cytochem 2004;52:931-42. |
|28.||Petit L, Maier E, Gibert M, Popoff MR, Benz R. Clostridium perfringens epsilon toxin induces a rapid change of cell membrane permeability to ions and forms channels in artificial lipid bilayers. J Biol Chem 2001;276:15736-40. |
|29.||Knapp O, Stiles B, Popoff MR. The Aerolysin-Like toxin family of cytolytic, pore-forming toxins. Toxinol J 2010;3:53-68. |
|30.||Al-Tahami K, Singh J. Smart polymer based delivery systems for peptides and proteins. Recent Pat Drug Deliv Formul 2007;1:65-71. |
|31.||Acharya S, Sahoo SK. PLGA nanoparticles containing various anticancer agents and tumour delivery by EPR effect. Adv Drug Deliv Rev 2011;63:170-83. |
|32.||Cryan SA. Carrier-based strategies for targeting protein and peptide drugs to the lungs. AAPS J 2005;7:E20-41. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]