|Year : 2019 | Volume
| Issue : 1 | Page : 21-24
Optimization of scaffolds for localized drug delivery: An In vitro study
Archana A Gupta1, Supriya Kheur1, Ravindra V Badhe2, Anagha Godse2, Ankita Shinkar2, A Thirumal Raj3
1 Department of Oral Pathology and Microbiology, Dr. D.Y. Patil Dental College and Hospital, Dr. D.Y. Patil Vidyapeeth, Pune, Maharashtra, India
2 Department of Pharmaceutical Sciences and Research, Dr. D.Y. Patil College of Pharmacy, Dr. D.Y. Patil Vidyapeeth, Pune, Maharashtra, India
3 Department of Oral Pathology and Microbiology, Sri Venkateswara Dental College and Hospital, Chennai, Tamil Nadu, India
|Date of Web Publication||22-Aug-2019|
Archana A Gupta
Department of Oral Pathology and Microbiology, Dr. D.Y. Patil Dental College and Hospital, Dr. D.Y. Patil Vidyapeeth, Pune - 411 041, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: In oncology, major surgical interventions are often followed by an interim period of 3–6 months allowing the patient to recover, following which conventional radiotherapy and chemotherapy are initiated. Lack of therapeutic interventions in the interim period allows proliferation of the residual tumor cells. Thus, introducing a treatment modality in the interim period which inhibits tumor cells without exuberating the postsurgical morbidity is the need of the hour. The objective of the study is to fabricate biodegradable cross-linked scaffolds incorporated with an anticancer drug and optimization of scaffolds for drug release. Materials and Methods: Qualitative and quantitative characterization of the drug was done with the help of high-performance liquid chromatography (HPLC) and ultraviolet (UV) analysis. Three-dimensional (3D) discs of biodegradable scaffolds were prepared with anticancer drugs in aqueous solution in different concentrations along with crosslinkers. Discs were studied for their release kinetics with the help of HPLC. Results: HPLC analysis of the 3D-discs revealed negative results. There was no sign of cisplatin absorbance after the scaffold immersion in the solution. The results were attributed to the rapid degradation of the drug. Conclusions: Although scaffold-mediated local chemotherapy holds a great potential to replace conventional chemotherapy as a postsurgical treatment modality, several practical limitations need to be addressed. Modification in the research methodology including a shorter time for preparing the scaffold and freeze-drying the scaffold material using lyophilization instead of normal drying could prevent degradation of the drug.
Keywords: Cancer, cisplatin, local chemotherapy, scaffold
|How to cite this article:|
Gupta AA, Kheur S, Badhe RV, Godse A, Shinkar A, Raj A T. Optimization of scaffolds for localized drug delivery: An In vitro study. J Pharm Negative Results 2019;10:21-4
|How to cite this URL:|
Gupta AA, Kheur S, Badhe RV, Godse A, Shinkar A, Raj A T. Optimization of scaffolds for localized drug delivery: An In vitro study. J Pharm Negative Results [serial online] 2019 [cited 2019 Nov 12];10:21-4. Available from: http://www.pnrjournal.com/text.asp?2019/10/1/21/265149
| Introduction|| |
Locoregional recurrence is a major cause for the poor survival rate in oral squamous cell carcinoma.,, 86% of recurrences occur within 24 months of postsurgical interventions., Thus, it is essential to revisit the current postsurgical treatment protocols to understand their limitations. At present, conventional chemotherapy and radiotherapy are the major forms of postsurgical interventions in oncology. The major limitation of conventional chemotherapy is the associated morbidity. The high dosage required in chemotherapy can be attributed to the lack of efficiency in the drug delivery system. The chemotherapeutic drugs undergo rapid glomerular filtration. Thus, for sufficient chemotherapeutic action, it is essential to increase both the dosage and duration of chemotherapy which in turn increases the systemic cytotoxicity. To overcome these limitations, recent studies have explored the use of targeted delivery systems wherein the effect of the chemotherapeutic agents is localized which increases the effectiveness of the therapeutic agent at a relatively lower dosage., Several means of delivery systems have been suggested for local chemotherapy including nanoparticles and scaffolds. Progress in tissue engineering has led to the emergence of a variety of scaffold biomaterials which are both biodegradable and biocompatible. Most of the scaffold-mediated research is based on common chemotherapeutic agents such as cisplatin. Several formulations of cisplatin have been studied with various scaffold materials including a biodegradable block copolymer, poly-cyclodextrin functionalized porous bioceramics, and bioresorbable polymersomes.,, Nanoparticle formulations of cisplatin with chitosan and that of paclitaxel have been extensively studied for effective drug release., Cytotoxic activity of cetuximab-modified silica nanoparticles and gold nanoparticles has also been explored in epidermal growth factor receptor-mutant lung cancer., The major limitation in scaffold-mediated local chemotherapy (SMLC) is the lack of optimal delivery of the drug for the required period at the required dosage. The major reason for this limitation is the lack of sufficient analytical investigation into drug formulations and its subsequent scaffold-mediated delivery on to the targeted site. Thus, in the present study, biodegradable scaffold material chitosan has been prepared and integrated with the anticancer drug cisplatin. The second stage of the study included qualitative and quantitative characterization of drug using ultraviolet (UV) spectrometry and high-performance liquid chromatography (HPLC), following which the scaffold-mediated drug release could be evaluated. The information from the present study could provide us with much insight into the practical application and current limitation in SMLC.
| Materials and Methods|| |
Instrument and apparatus: A double-beam UV spectrophotometer with a spectral width of 2 nm, Shimadzu Model 1800 (Japan), was used. The wavelength accuracy of the instrument was 0.5 nm fitted with a pair of 10 mm matched quartz cell to measure the absorbance of the serial dilution solutions.
Cisplatin was taken in solution form. A stock solution of 0.9% sodium chloride was prepared by mixing 0.9 g of sodium chloride in 100 mL of distilled water. A volume of 5 mL of cisplatin drug in the solution form was mixed in 50 mL of stock solution to make the concentration of drug adjusted to 5 μg/mL of stock solution. Serial dilutions of this drug solution were made as 0.5, 1, 2, 3, 4, 5, and 6 mL with the help of 2 mL pipette taking the lower meniscus. The volume was adjusted with the help of stock solution.
The working standard solutions were scanned in the UV-Visible range of 400–800 nm, using the prepared stock solution as blank for obtaining the absorption maxima (λ max). A calibration curve was performed to measure the linearity of the curve in the concentration range using serial dilutions of the solution. Measurements were taken on samples prepared on 3 consecutive days (n =3). The values were reported as the mean ± confidence interval of the calibration curves. The data were analyzed at a wavelength of 254 nm.
High-performance liquid chromatography
Instrument and apparatus included a HPLC system consisting of an HPLC Agilent 1100 Series Diode array and multiple Wavelength Detectors, a rhenodyne injector fitted with a 100 μl sample loop, and an RP 18 end-capped Purospher ® STAR Merck column (250 mm × 4.6 mm, 5 μm p) and guard column (4 mm × 3 mm, 5 μm, Hichrom, Kromasil); the mobile phase flow rate was maintained 1 mL/min. The method was validated in accordance with the US Pharmacopoeia and the International Conference on Harmonization. Serial dilutions of 10, 15, and 20 μL of the drug in stock solution were prepared. The mobile phase used is 80:20 ACN: Methanol. Qualitative and quantitative detection of the drug was made with the help of HPLC.
The hydrogel of 2% chitosan with the 5 mL drug was prepared with crosslinkers TPP in increasing concentrations of 5%, 10%, 15%, and 20%. The solution was poured in 24 well plates and kept in an incubator at 37°C for drying. Dried scaffolds were then placed in distilled water for drug release experiment.
| Results|| |
Analytical curves were obtained on 3 consecutive days (n =3). The mean of absorbance was plotted at 706 nm against the concentration. The curves were found to be linear for serial dilutions of the drug in the concentration of 5 μg/mL of drug and yielded a correlation coefficient (r) of 0.9738 [Figure 1].
|Figure 1: Linearity curve for increased absorbance with an increased concentration of drug|
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High-performance liquid chromatography analysis
Linearity curve of cisplatin was obtained with increasing concentrations of drug for 25 μL and 50 μL after 12 h. There was no increase in area as the drug concentration increased as shown in [Figure 2] and [Figure 3].
The presence of the drug in the solution was analyzed with the help of HPLC after 24 h. The results obtained revealed only one peak as shown in [Figure 4]. Furthermore, it yielded tailing of peak along with the absence of a straight baseline.
|Figure 4: No straight baseline and tailing of the peak of absorption spectra of cisplatin|
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Drug release experiments
The scaffold material was placed in a phosphate buffer for 24 h, 48 h, and 72 h to have the drug leached out of the scaffold material. The phosphate buffer solution was then analyzed spectrophotometrically under UV analyzer. However, no peak was observed corresponding to cisplatin absorbance.
| Discussion|| |
UV spectrophotometer quantitative analysis of the drug showed a linear curve of increased absorbance with increased concentration of the drug. This helped to validate that the solution form of the drug to be used in experiments contain an appropriate amount of cisplatin required to make the scaffolds. The absorbance rate of the drug also increased with the increase in concentration giving a linear curve. However, when the same solution was tested quantitatively for HPLC analysis after 12 h, the area covered by the curve did not show a linear increase with the increased concentration of the drug. It can be explained on the fact that UV analysis may not be an appropriate method for characterization of cisplatin in the solution form. The qualitative characterization of the drug solution was done with the help of HPLC after 24 h. The curve did not reveal any peak corresponding to cisplatin absorbance. Furthermore, the absorbance curve showed the absence of a straight baseline along with its tailing suggesting the degradation of the drug once the solution is opened. The experiments were proceeded further for scaffold preparation by incorporating the crosslinkers and drug solution in the biodegradable scaffold material solution. Dried scaffolds were analyzed for drug release experiments, but the phosphate buffer did not reveal any peak pertaining to cisplatin absorbance after the scaffold immersion in the solution. This can be explained on the grounds of the degraded drug which did not give expected results under HPLC and further gave negative results for drug release experiments in UV analysis. In addition, due to wet weather conditions, the scaffolds took a longer duration to get dried under ambient room temperature. This may also be the reason for the degradation of the drug. The results revealed major limitation in our present methodology. As the drug in solution form degrades faster, it is vital to shorten the scaffold preparation time. In addition to this, it is also important to avoid normal drying of the scaffold material. Instead, the scaffold material should be lyophilized wherein the scaffolds will be freeze-dried as soon as they are formed.
| Conclusions|| |
The morbidity associated with conventional chemotherapy has led to the search for localized chemotherapy delivery systems. Recent studies on SMLC have shown success in limiting the proliferation of cancer cells at a relative minimal dosage. The present study was successful in formulating a biodegradable cross-linked scaffold, but the anticancer drug cisplatin which was integrated into the scaffold showed rapid degradation. Thus, to conclude, although SMLC has the potential to replace conventional chemotherapy as the postsurgical treatment of choice in cancer, further research is required to stabilize the drug and to optimize the drug delivery dosage and duration.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Agra IM, Filho JG, Martins EP, Kowalski LP. Second salvage surgery for re-recurrent oral cavity and oropharynx carcinoma. Head Neck 2010;32:997-1002.
Leemans CR, Tiwari R, Nauta JJ, van der Waal I, Snow GB. Recurrence at the primary site in head and neck cancer and the significance of neck lymph node metastases as a prognostic factor. Cancer 1994;73:187-90.
Carvalho AL, Kowalski LP, Agra IM, Pontes E, Campos OD, Pellizzon AC. Treatment results on advanced neck metastasis (N3) from head and neck squamous carcinoma. Otolaryngol Head Neck Surg 2005;132:862-8.
Fan S, Tang QL, Lin YJ, Chen WL, Li JS, Huang ZQ, et al.
Areview of clinical and histological parameters associated with contralateral neck metastases in oral squamous cell carcinoma. Int J Oral Sci 2011;3:180-91.
Kowalski LP, Carvalho AL, Martins Priante AV, Magrin J. Predictive factors for distant metastasis from oral and oropharyngeal squamous cell carcinoma. Oral Oncol 2005;41:534-41.
Vermorken JB, Remenar E, van Herpen C, Gorlia T, Mesia R, Degardin M, et al.
Cisplatin, fluorouracil, and docetaxel in unresectable head and neck cancer. N
Engl J Med 2007;357:1695-704.
Kartalou M, Essigmann JM. Mechanisms of resistance to cisplatin. Mutat Res 2001;478:23-43.
Pinzani V, Bressolle F, Haug IJ, Galtier M, Blayac JP, Balmès P, et al.
Cisplatin-induced renal toxicity and toxicity-modulating strategies: A review. Cancer Chemother Pharmacol 1994;35:1-9.
Chai F, Abdelkarim M, Laurent T, Tabary N, Degoutin S, Simon N, et al.
Poly-cyclodextrin functionalized porous bioceramics for local chemotherapy and anticancer bone reconstruction. J Biomed Mater Res B Appl Biomater 2014;102:1130-9.
Petersen MA, Hillmyer MA, Kokkoli E. Bioresorbable polymersomes for targeted delivery of cisplatin. Bioconjug Chem 2013;24:533-43.
Surnar B, Subash PP, Jayakannan M. Biodegradable block copolymer scaffolds for loading and delivering cisplatin anticancer drug. Anorg Allg Chem 2014;640:1119-26.
Kang RH, Kwon JY, Kim Y, Lee SM. Cisplatin-mediated formation of polyampholytic chitosan nanoparticles with attenuated viscosity and pH-sensitive drug release. Langmuir 2017;33:9091-9.
Ma P, Mumper RJ. Paclitaxel nano-delivery systems: A Comprehensive review. J Nanomed Nanotechnol 2013;4:1000164.
Wang Y, Huang HY, Yang L, Zhang Z, Ji H. Cetuximab-modified mesoporous silica nano-medicine specifically targets EGFR-mutant lung cancer and overcomes drug resistance. Sci Rep 2016;6:25468.
Qian Y, Qiu M, Wu Q, Tian Y, Zhang Y, Gu N, et al.
Enhanced cytotoxic activity of cetuximab in EGFR-positive lung cancer by conjugating with gold nanoparticles. Sci Rep 2014;4:7490.
Shabir GA. Validation of high-performance liquid chromatography methods for pharmaceutical analysis. Understanding the differences and similarities between validation requirements of the US food and drug administration, the US pharmacopeia and the international conference on harmonization. J Chromatogr A 2003;987:57-66.
Tezcana S, Özdemirb F, Turhalc S, İzzettina FV. High performance liquid chromatographic determination of free cisplatin in different cancer types. Pharm Chem 2013;5:169-74.
Basotra M, Singh SK, Gulati M. Development and validation of a simple and sensitive spectrometric method for estimation of cisplatin hydrochloride in tablet dosage forms: Application to dissolution studies. ISRN Anal Chem 2013. p. 1-8.
Haugh MG, Murphy CM, O'Brien FJ. Novel freeze-drying methods to produce a range of collagen-glycosaminoglycan scaffolds with tailored mean pore sizes. Tissue Eng Part C Methods 2010;16:887-94.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]