Formulation of nanoparticles from short chain chitosan as gene delivery system and transfection against T47D cell line

Recently numerous prototype DNA-based biopharmaceuticals can be used to  control  disease  progression  by  induction  and  inhibitin  the  overexpression  of genes.  Since  there  are  poor  cellular  uptake  and  rapid  in  vivo  degradation  of DNA-based  therapeutics  therefore  the  use  of  delivery  systems  to  facilitate cellular internalization and preserve their activity is necessary. Cationic polymers commonly used as carriers to delivery gene because of easy to form complexes and  higher  stability  compared  to  that  lipoplexs.  Chitosan,  a  cationic,  are polymer most widely used in gene delivery systems because of the low toxicity, and biocompatible. The aim of this study was to formulate nanoparticles of short chain  chitosan-pEGFP-C1  and  short  chain  chitosan/TPP-pEGFP-C1  by coaservation  complex  method.  Stability  test  of  the  formula  was  performed  by incubating the nanoparticles complex with DNase I and Artificial Intestinal Fluid. Cytotoxicity  and transfection  studies  were  evaluated  against  T47D  cell line.  The diameter  of  Chitosan-pEGFP-C1  and  chitosan/TPP-pEGFP-C1  nanoparticles  were on the range of 56–282.8 nm. The zeta potential wasdetermined to be +14.03 - +16.6  mV.  Stability  studies  showed  that  chitosan-pEGFP-C1  and  chitosan/TPPpEGFP-C1  nanoparticles  were  stable,  undegradable  by  DNase  I  and  artificial intestinal fluid. Cytotoxic Assay of Chitosan-pEGFP-C1 and  chitosan/TPP-pEGFPC1  nanoparticles  (pH  4.0)  showed  that  the  viability  of cell  was  >  90%  for  all formulas.  EGFP-C1  plasmid  gene  delivered  by  chitosan  nanoparticles  can  be expressed  in  T47D  cell  culture.  According  to  these  results  chitosan  and chitosan/TPP  nanoparticles  had  potentially  to  be  used  as  a  non-viral  vector system delivery for gene therapy.Key words:Chitosan, Nanoparticles, Plasmid EGFP-C1, Cell culture T47D

CHITOSAN NANOPARTICLE AS A DELIVERY SYSTEM FOR POLYPHENOLS FROM MENIRAN EXTRACT (PHYLLANTHUS NIRURI L.): FORMULATION, OPTIMIZATION, AND IMMUNOMODULATORY ACTIVITY

Objective: This study aims to formulate meniran extract into polymeric nanoparticles. Better stability of active substances in formulas compared to unformulated extracts is expected to increase immunomodulatory activity. Methods: Nanoparticles were formulated using ionic gelation method with chitosan and tripolyphosphate polymers. Optimize the mixture of nanoparticles using simplex lattice design (SLD) with the help of Design-Expert (DX) software. Evaluation of particle size and potential zeta using dynamic light scattering (DLS). Interactions between components were analyzed using Fourier transform infrared spectrophotometry-attenuated total reflectance (FTIR-ATR) and morphology of the lyophilization results observed using scanning electron microscopy (SEM). Immunomodulatory tests using the latex assay method. The parameters tested included phagocytosis index, phagocytic activity, and nitric oxide secretion. Results: The optimum mixture of the formulation process was obtained in the composition of chitosan 0.270 %, extract 0.626 %, and tripolyphosphate 0.074 % with desirability value of 0.841. Optimal response with particle size 434.7±3.90 d. nm, polydispersity index 0.285±0.03 and entrapment efficiency 62.98±0.65 %. The zeta potential value in the optimum formula is 11.9±0.1 mV with a positive charge. Phagocytosis index and phagocytic activity of nanoparticles differed significantly (p<0.05) compared with unformulated extracts. Conclusion: Meniran extract was successfully formulated into polymeric nanoparticles using chitosan-tripolyphosphate polymer. The developed nanoparticles have the immunomodulatory activity that is better than unformulated extract

FORMULATION OF NANOCURCUMIN USING LOW VISCOSITY CHITOSAN POLYMERAND ITS CELLULARUPTAKE STUDY INTO T47D CELLS

Using of curcumin as anti cancer agent is restricted by its low solubility, therefore it has low bioavability. This obstacle can be solved by the development of curcumin nanoparticle. Nanoparticle technology has been started to be developed as an alternative solution to improve drug delivery pofile, especially for the less bio-available chemical. This study was aimed to develope nanocurcumin formulation with low viscosity chitosan as the matrix and to study its ability to be taken into the cells in vitro. Method used in the formulation of nanocurcumin in this study is by ionic gelation followed by freeze drying. Entrapment Efficiency then assayed, and its stability was tested by incubating the formula into artificial intestinal fluid (AIF). Furthermore, its toxicity was evaluated, also its cellular uptake ability into T47D cell line. It was found that the Entrapment Efficiency in acetate buffer at pH 4 is higher than at pH 5. This formula also has a good stability in AIF. For the cellular uptake study through fluorescence microscope, it was found that the complex has an ability to penetrate cellular membrane into the cytosol. The cytotoxicity study tell us that the nanocurcumin is non-toxic to normal cell line. For the characterization of the nanoparticles, the average size of this particle is 269.8 nm, its zeta-potential is +18.63 mV, with spherical particle morphology. From the result of this study, it is concluded that formulation of nanocurtumin using low viscosity chitosan polymer as the matrix has a great potential as an alternative for anticancer therapy

FORMULATION OF NANOCURCUMIN USING LOW VISCOSITY CHITOSAN POLYMER AND ITS CELLULAR UPTAKE STUDY INTO T47D CELLS

Using  of  curcumin  as  anti  cancer  agent  is  restricted  by  its low  solubility,  therefore  it  has  low  bioavability.  This  obstacle  can be  solved  by  the  development  of  curcumin  nanoparticle. Nanoparticle  technology  has  been  started  to  be  developed  as  an alternative  solution to  improve drug  delivery pofile, especially  for the less bio-available chemical. This study was aimed to develope nanocurcumin  formulation  with  low  viscosity  chitosan  as  the matrix  and  to  study  its  ability  to  be  taken  into  the cells in  vitro. Method  used  in  the  formulation  of  nanocurcumin  in  this  study  is by ionic gelation followed by freeze drying. Entrapment  Efficiency then  assayed,  and  its  stability  was  tested  by  incubating  the formula  into  artificial  intestinal  fluid  (AIF).  Furthermore,  its toxicity  was  evaluated,  also  its  cellular  uptake  ability  into  T47D cell  line.  It  was  found  that  the  Entrapment  Efficiency  in  acetate buffer  at  pH  4  is  higher  than  at  pH  5.  This  formula  also  has  a good  stability  in  AIF.  For  the  cellular  uptake  study  through fluorescence  microscope,  it  was  found  that  the  complex  has  an ability  to  penetrate  cellular  membrane  into  the  cytosol.  The cytotoxicity  study  tell  us  that  the  nanocurcumin  is  non-toxic  to normal  cell line. For  the characterization of the nanoparticles, the average  size  of  this  particle  is  269.8  nm,  its  zeta-potential  is +18.63 mV, with spherical particle morphology. From the result ofthis study, it is concluded that formulation of nanocurcumin using low viscosity chitosan polymer as the matrix has a great potential as an alternative for anticancer therapy.Key words: nanoparticle, curcumin, low viscosity chitosan, T47D cell line.

Pengaruh Propilen Glikol, Asam Oleat, Dan Isopropilalkohol Pada Formula Patch Transdermal Kalium Losartan

Losartan merupakan obat antihipertensi poten dengan bioavailabilitas rendah dan waktu paruh eliminasi cepat. Penelitian ini bertujuan untuk mengetahui komposisi formula optimum, karakteristik dan profil transpor  in vitro  patch transdermal kalium losartan. Rancangan formula berdasarkan metode simplex lattice design menggunakan software Design Expert. Evaluasi  karakteristik meliputi  ketebalan, bobot, moisture uptake, loss on drying,  folding enduranc,  dan drug content. Uji transport in vitro menggunakan  sel difusi vertikal, penetapan kadar transpor losartan menggunakan instumen HPLC dan analisis data menggunakan software WinSAAM. Komposisi formula optimum patch transdermal  losartan adalah 44,4% propilen glikol, 29,3% asam oleat,  dan 26,3% isopropil alkohol,  dengan karakteristik  tebal 0,6 mm, bobot 82,2 mg, loss on drying12,8%, moisture uptake 6,4%, folding endurance 300 lipatan dan drug content 98,9%. Profil transpor in vitro losartan menghasilkan model lima kompartemen dengan kinetika orde pertama

FORMULATION OF SELF-NANOEMULSIFYING DRUG DELIVERY SYSTEM OF BOVINE SERUM ALBUMIN USING HLB (HYDROPHILIC-LYPOPHILIC BALANCE) APPROACH

Self-Nanoemulsifying Drug Delivery System (SNEDDS) has potential to be developed for oral protein delivery because it is free from water, hence preserving the stability of protein, protecting protein from enzymatic degradation, and enhancing the protein permeability in the gastrointestinal tract (GIT). However, protein-based SNEDDS formulation is challenging due to low solubility property of protein in oil, which is towards zero. This present study aimed to obtain the most compatible SNEDDS system for protein using HLB approach. Bovine serum albumin (BSA) was used as a protein model in this study. A number of 78 formulae with HLB ranging between 11 and 15 were screened to acquire stable SNEDDS composition without the presence of phase separation. Of 13 stable formulae, two were selected (F30, F45) with HLB 15, and then loaded with BSA. Physical characteristics of both formulae were then evaluated and the results suggested that SNEDDS with single hydrophilic surfactant (F45) and HLB 15 was the best formula for protein template as the stability testing showed that phase separation and precipitation did not appear. It was robust to pH and dilution with percentage of transmittance of 96.40±1.05% and the droplet size of 180.9nm. F45 also had uniform distribution of droplets size since the polydispersity index was less than 0.1. The zeta potential of F45 was -0.12mv with loading efficiency 83.57±1.77%. The emulsifying time of F45 was > 2min due to the formation of crystalline gel that was difficult to disperse

Pengaruh Propilen Glikol, Asam Oleat, Dan Isopropilalkohol Pada Formula Patch Transdermal Kalium Losartan

Losartan merupakan obat antihipertensi poten dengan bioavailabilitas rendah dan waktu paruh eliminasi cepat. Penelitian ini bertujuan untuk mengetahui komposisi formula optimum, karakteristik dan profil transpor  in vitro  patch transdermal kalium losartan. Rancangan formula berdasarkan metode simplex lattice design menggunakan software Design Expert. Evaluasi  karakteristik meliputi  ketebalan, bobot, moisture uptake, loss on drying,  folding enduranc,  dan drug content. Uji transport in vitro menggunakan  sel difusi vertikal, penetapan kadar transpor losartan menggunakan instumen HPLC dan analisis data menggunakan software WinSAAM. Komposisi formula optimum patch transdermal  losartan adalah 44,4% propilen glikol, 29,3% asam oleat,  dan 26,3% isopropil alkohol,  dengan karakteristik  tebal 0,6 mm, bobot 82,2 mg, loss on drying12,8%, moisture uptake 6,4%, folding endurance 300 lipatan dan drug content 98,9%. Profil transpor in vitro losartan menghasilkan model lima kompartemen dengan kinetika orde pertama

FORMULATION AND CHARACTERIZATION OF TIMOLOL MALEATE-LOADED NANOPARTICLES GEL BY IONIC GELATION METHOD USING CHITOSAN AND SODIUM ALGINATE

Objective: The objectives of this study were to formulate and characterize nanoparticles gel of timolol maleate (TM) by ionic gelation method using chitosan (CS) and sodium alginate (SA). Methods: Optimization was carried out by factorial design using Design Expert®10.0.1 software to obtain the concentration of CS, SA, and calcium chloride (CaCl2) to produce the optimum formula of TM nanoparticles. The optimum formula was characterized for particle size, polydispersity index, entrapment efficiency, Zeta potential, and molecular structure. Hydroxy Propyl Methyl Cellulose (HPMC) K15 was incorporated into optimum formula to form nanoparticles gel of TM and carried out in vivo release study using the Franz Diffusion Cell. Results: TM nanoparticles was successfully prepared with concentration of CS, SA, and CaCl2 of 0.01 % (w/v), 0.1 % (w/v), and 0.25 % (w/v), respectively. The particle size, polydispersity index, entrapment efficiency, and Zeta potential were found to be 200.47±4.20 nm, 0.27±0.0154, 35.23±4.55 %, and-5.68±1.80 mV, respectively. The result of FTIR spectra indicated TM-loaded in the nanoparticles system. In vitro release profile of TM-loaded nanoparticles gel showed controlled release and the Korsmeyer-Peppas model was found to be the best fit for drug release kinetics. Conclusion: TM-loaded CS/SA nanoparticles gel was successfully prepared and could be considered as a promising candidate for controlled TM delivery of infantile hemangioma treatment

Formulation of Insulin Self Nanoemulsifying Drug Delivery System and Its In Vitro-In Vivo Study

Particulate delivery system can be used for improving the efficacy of protein and peptide drug. In addition to a polymer-based particulate delivery system, self-nanoemulsifying drug delivery system (SNEDDS), a lipid-based delivery system, is currently developed for either less water-soluble or soluble drugs. This study aims to design SNEDDS for oral insulin administration and its in vitro-in vivo study. The SNEDDS template was designed using D-optimal mixture design and was analyzed using software Design Expert 7.1.5. The obtained optimum template was loaded with insulin and evaluated for its transmittance percentage, emulsification time, particle size, zeta potential, stability, the amount of insulin in vitro diffused across rat intestine, and insulin serum concentration after oral administration. The study results revealed that the optimum template of SNEDDS formula consisted of 10% (w/w) Miglyol 812N, 65% (w/w) Tween 80, and 25% (w/w) propylene glycol. These optimum template then was loaded with insulin and characterized. SNEDDS insulin has particle size of 12.0±1.7 nm, zeta potential of +0.16mV, transmittance of >90%, and emulsification time of < 60 seconds. The stability study showed that SNEDDS insulin was stable from both precipitation and phase separation. The amount of insulin transported from SNEDDS formula in vitro was 32.45±2.03% and non-SNEDDS formula was 10.44±5.04%. In vivo study of SNEDDS insulin produced a significantly increased Cmax, AUC, and F value than insulin non SNEDDS (p < 0.05). In brief, SNEDDS formulation in this study is a promising approach to increase the effectiveness of oral insulin. Insulin is better given orally in SNEDDS formulation than in non SNEDDS formulation

CHITOSAN NANOPARTICLES MEDIATED DELIVERY OF MIR-106B-5P TO BREAST CANCER CELL LINES MCF-7 AND T47D

Objective: The development of nanomedicine, such as miRNA transfection to cancer cells,has widely gained interest in the past decade. Unfortunately, miRNA tends to decay easily by the cellular enzymatic process and requires a carrier. As a cationic biopolymer, chitosan is widely known as a non-viral vector. However, research about chitosan as a miRNA delivery system remains limited. This study aimed to investigate the effect and characters of synthetic miRNA loaded chitosan nanoparticles on breast cancer cell lines. Methods: To obtain the nanocomplex, chitosan-antimiR-106b-5p was formulated using natriumtripolyphosphate through ionic gelation methods. The nanochitosan formula was characterized by using gel electrophoresis; Nano Quant for encapsulation of entrapment quantification; morphology appearance as viewed by Scanning Electron Microscope (SEM), nanochitosan size analysis; in vitro analysis using MCF-7 and T47D breast cancer cell lines; in silico prediction of possible gene target; polymerase chain reaction analysis and gel electrophoresis for E2F1/GAPDH expression. Results: The efficiency entrapment value was 96.7%, particle size analysis was 458±11.79 nm, and polydispersity index (PDI) was 0.65±0.07, with spherical morphology as viewed in SEM. There was no significant difference between the nanochitosan supplemented group and the control group in MCF-7 cells (p=0.067). However, the ratio of E2F1 to GAPDH was significantly lower than the control group after nanochitosan antimiR-106b-5p was loaded at concentration 140 nmol (p=0.022) and 35 nmol (p=0.016). Conclusion: Our nanochitosan formula is non-toxic to use in MCF-7 cell lines. Most importantly, as the formula was conjugated to synthetic antimiR-106b-5p, the E2F1 expression decreased