Research progress of in vitro-in vivo correlation of injectable polylactide-polyglycolide microspheres / 药学学报

As a depot drug delivery system, injectable polylactide-polyglycolide (PLGA) sustained-release microspheres have been successfully used to treat many diseases since the first microsphere product Lupron depot was approved for marketing in the United States in 1989. It has the ability of long-term release in the body for several days to several months, which can not only reduce the times of administration, but also reduce the drug blood concentration fluctuations, significantly improve the safety and patient compliance. In vitro-in vivo correlation (IVIVC) makes the development of microspheres more possible. It can describe the dynamic information of drug release in vivo through the in vitro release behavior of microspheres, and can reduce the workload of each stage and shorten the time span while characterizing the performance of microspheres. IVIVC can provide guidance or support for drug development, production changes, supervision and management. This article summarizes the release mechanism of injectable PLGA sustained-release microspheres, common measurement methods and theories of in vitro and in vivo release. And we also focus on the establishment and application of IVIVC, especially A level IVIVC in the field of microsphere preparations, to provide a reference for further study on in vitro-in vivo correlation of microspheres.

Preparation and in vitro release profiling of PLGA microspheres containing BSA as a model protein

Conventional drug formulations are incapable of adequate delivery of proteins and peptides for therapeutic purposes. As these molecules have very short biological half-life, multiple dosing is required to achieve the desirable therapeutic effects. Microspheres are able to encapsulate proteins and peptide in the polymeric matrix while protecting them from enzymatic degradation. In this study Bovine Serum Albumin (BSA) matrix type microspheres were fabricated using Polylactide-co-glycolide (PLGA) by double emulsion solvent evaporation method. The effects of variables such as homogenizer speed, molecular weight of polymer and the effect of pH of the water phases, were investigated against factors such as drug loading, encapsulation efficiency, morphology, size, drug distribution and release profile of the microspheres. Results, suggested that an increase in homogenization speed leads to a decrease in microsphere size. The increase in homogenization speed also caused a significant effect on the release profile only when higher molecular weight of polymer had been used.. The pH change of the internal aqueous phase led to modification of surface morphology of spheres to a porous structure that significantly increased the total amount of released protein. Integrity of protein structure was intact as shown by SDS-PAGE. According to the results, it can be concluded that we achieved a reproducible method regarding controlled protein delivery for different sizes of particles.

Vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor mixed microcapsules in promoting angiogenic differentiation of rat bone marrow mesenchymal stem cells in vitro / 中国修复重建外科杂志

Objective: To observe the effect of vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor (VEGF/PELA/bFGF) mixed microcapsules in promoting the angiogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods: The BMSCs were isolated by the method of whole bone marrow adherent, and sub-cultured. The passage 3 BMSCs were identified by Wright-Giemsa staining and flow cytometry, and used for subsequent experiments. VEGF/PELA/bFGF (group A), PELA/bFGF (group B), VEGF/PELA (group C), and PELA (group D) microcapsules were prepared. The biodegradable ability and cytotoxicity of PELA microcapsule were determined，and the slow-released ability of VEGF/PELA/bFGF mixed microcapsules was measured. The passage 3 BMSCs were co-cultured with the extracts of groups A, B, C, and D, separately. At 1, 3, 7, 14, and 20 days after being cultured, the morphological changes of induced BMSCs were recorded. At 21 days, the induced BMSCs were tested for DiI-labeled acetylated low density lipoprotein (Dil-ac-LDL) and FITC-labeled ulex europaeus agglutinin I (FITC-UEA-I) uptake ability. The tube-forming ability of the induced cells on Matrigel was also verified. The differences of the vascularize indexes in nodes, master junctions, master segments, and tot.master segments length in 4 groups were summarized and analyzed. Results: The isolated and cultured cells were identified as BMSCs. The degradation time of PELA was more than 20 days. There was no significant effect on cell viability under co-culture conditions. At 20 days, the cumulative release of VEGF in the mixed microcapsules exceeded 95%, and the quantity of bFGF exceeded 80%. The morphology of cells in groups A, B, and C were changed. The cells in groups A and B showed the typical change of cobble-stone morphology. The numbers of double fluorescent labeled cells observed by fluorescence microscope were the most in group A, and decreases from group B and group C, with the lowest in group D. The cells in groups A and B formed a grid-like structure on Matrigel. Quantitative analysis showed that the differences in the number of nodes, master junctions, master segments, and tot.master segments length between groups A, B and groups C, D were significant ( P0.05). Conclusion: VEGF/PELA/bFGF mixed microcapsules have significantly ability to promote the angiogenic differentiation of rat BMSCs in vitro.

Recovery effect of PLGA microsphere-loaded VAP combined with BMMSCs on sciatic nerve injury of rats and its mechanism / 吉林大学学报(医学版)

Objective: To observe the recovery effect of polylactide-glycolic acid (PLGA) microsphere-loaded velvet antler peptide (VAP) combined with bone marrow mesenchymal stem cells (BMMSCs) on the sciatic nerve injury of the rats, and to explore the related mechanisms. Methods: The sciatic nerve injury models were made by the cross-sectional method. A total of 60 rats were randomly divided into sciatic nerve transection control group (control group), VAP group, BMMSCs transplantation (BMC) group and VAP microsphere combined with BMMSCs transplantation (VAP-BMC) group (n=15). Seven days after modeling, the rats in control group were injected with PBS solution in the transecting sciatic nerve region, the rats in VAP group were injected with PBS solution containing PLGA microspheres loading drug, the rats in BMC group were injected with PBS solution containing BMMSCs, and the rats in VAP-BMC group were injected with PBS solution containing PLGA microspheres loading drug and BMMSCs. After three months, the neurological function of the rats in various groups were evaluated by Basso, Beattie, Bresnahan (BBB) functional sports rating scale, and the morphology of sciatic nerves of the rats in various groups were detected by HE staining. The expression levels of protein disulfide isomerase (PDI), glucose regulated protein-78 (GRP-78) and Caspase-12 in sciatic nerve of the rats in various groups were detected by Western blotting method. Results; The results of BBB functional sports rating scale showed that compared with control group, the BBB scores of the rats in VAP group, BMC group and VAP-BMC group were increased (P<0. 01), especially in VAP-BMC group. The results of HE staining showed that compared with control group, the sciatic nerve fibers of the rats in VAP group, BMC group and VAP-BMC group got thicker and the diameters of axon were longer (P<0. 01). The results of Western blotting showed that compared with control group, the expression levels of PDI and Caspase-12 in the sciatic nerve of the rats in VAP group, BMC group and VAP-BMC group were significantly decreased (P<0. 01), and the expression level of GRP-78 protein in the sciatic nerve of the rats in VAP-BMC group was significantly increased (P<0. 01). Conclusion: PLGA microsphere-loaded VAP combined with BMMSCs can significantly promote the repairment of injured sciatic nerves, and its mechanism may be related to the inhibition of endoplasmic reticulum stress.

Unsintered Hydroxyapatite and Poly-L-Lactide Composite Screws/Plates for Stabilizing β-Tricalcium Phosphate Bone Implants

Unsintered hydroxyapatite (u-HA) and poly-L-lactide (PLLA) composites (u-HA/PLLA) are osteoconductive and biodegradable. Screw (Super-Fixsorb MX30) and plate (Super-Fixsorb MX40 Mesh) systems made of u-HA/PLLA are typically used in small bones in maxillofacial surgeries. After the resection of bone tumors in larger bones, reconstructions with β-tricalcium phosphate (β-TCP) implants of strong compression resistance have been reported. After a resection, when the cavity is hemispheric- or concave-shaped, stabilization of the implanted β-TCP block is necessary. In the current series, u-HA/PLLA were used to stabilize the mechanically strong implanted low-porous β-TCP blocks in six bone tumor cases, including three giant cell tumors of bone, and one case each of chondroblastoma, chondrosarcoma, and parosteal osteosarcoma. The mean age of patients at the time of surgery was 31.3 years (range, 19 to 48 years). The bones involved were two ilia (posterior), a femur (diaphysis to distal metaphysis), and three tibias (proximal epiphysis, proximal metaphysis to epiphysis, and distal metaphysis to epiphysis). Neither displacement of the implanted β-TCP block nor any u-HA/PLLA-related complications were observed. The radiolucent character of the u-HA/PLLA did not hinder radiological examinations for potential signs of tumor recurrence. The method of using u-HA/PLLA components for the stabilization of β-TCP blocks makes the procedure easy to perform and reliable. It can extend the application of β-TCP blocks in reconstruction surgery.

Preparation and quality control of human anti-VEGFR-2/As2 O3-PEG-PLA nanoparticle / 重庆医学

Objective To explore the preparation and quality control of As2 O3 nanoparticle .Methods PEG‐PLA was used as the vector material to prepare As2 O3 nanoparticle with ultrasonic emulsification method ,and the VEGFR‐2 was coupled to obtain VEGFR‐2/As2 O3‐PEG‐PLA nanoparticle .The particle size distribution ,Zata potential ,loading efficiency (LE) ,encapsulation effi‐ciency(EE) ,drug release in vitro and stability was determined ,and morphological characteristics was observed by transmission elec‐tron microscope(TEM) .Tweety‐four hepatocellular carcinoma nude mices were randomly divided into VEGFR‐2/As2O3‐PEG‐PLA nanoparticles group and As2 O3‐PEG‐PLA nanoparticles group ,by tail vein injection of nanoparticles .High performance liquid chro‐matography was used to determine content of As2 O3 .After 21 d ,six nude mices in each group were killed ,and the immunohisto‐chemistry and western blot method was used to detect the expression of VEGFR‐2 .Results The particle size of VEGFR‐2/As2 O3‐PEG‐PLA was determined to be (141 .9 ± 13 .2)nm ,Zata potential was (10 .2 ± 1 .1)mV .It was found to spherical or oval shape , with uniform size and dispersibility under TEM .LE and EE was (5 .51 ± 1 .83)% and (62 .12 ± 5 .98)% ,respectively .Drug release in vitro showed that VEGFR‐2/As2 O3‐PEG‐PLA exhibited controlled release effect ,with half of the release time as 10 h .Besides , VEGFR‐2/As2 O3‐PEG‐PLA showed a good stability in 3 days .Compared with As2 O3‐PEG‐PLA nanoparticles group ,the concen‐tration of As2 O3 in tumor and liver tissue was high ,the concentration of As2 O3 in blood ,heart ,kidney tissue was low ,the expression of VEGFR‐2 in tumor tissue was low in VEGFR‐2/As2O3‐PEG‐PLA nanoparticles group(P< 0 .05) .Conclusion The prepared As2 O3 nanoparticle using PEG‐PLA as vector and VEGFR‐2 as target showed uniform size ,high EE and LE ,good stability .And it preliminarily proved that VEGFR‐2 could be targeted in nude mice .

Synthesis and characterization of polylactide-based thermosetting polyurethanes with shape memory properties / 生物工程学报

A series of bio-based thermosetting polyurethanes (Bio-PUs) were synthesized by the crosslinking reaction of polylactide and its copolymers diols with hexamethylene diisocyanate (HDI) trimer. The obtained Bio-PUs were characterized by Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), Thermal Gravimetric Analysis (TGA), universal tensile testing machine and cytotoxicity test. Results indicate that the PLA copolymer (P(LA-co-CL)) diols reduced the glass transition temperature (Tg) of Bio-PUs and improved their thermal stability, compared with PLA diols. The Bio-PUs synthesized from P (LA-co-CL) diols exhibit better mechanical performance and shape memory properties. Especially, Young modulus and elongation at break of the obtained Bio-PUs were 277.7 MPa and 230% respectively; the shape recovery time of the obtained Bio-PUs at body temperature was only 93 s. Furthermore, alamar blue assay results showed that the obtained Bio-PUs had no cell toxicity.

Nanocarrier-mediated co-delivery of chemotherapeutic drugs and gene agents for cancer treatment

The efficacy of chemotherapeutic drug in cancer treatment is often hampered by drug resistance of tumor cells, which is usually caused by abnormal gene expression. RNA interference mediated by siRNA and miRNA can selectively knock down the carcinogenic genes by targeting specific mRNAs. Therefore, combining chemotherapeutic drugs with gene agents could be a promising strategy for cancer therapy. Due to poor stability and solubility associated with gene agents and drugs, suitable protective carriers are needed and have been widely researched for the co-delivery. In this review, we summarize the most commonly used nanocarriers for co-delivery of chemotherapeutic drugs and gene agents, as well as the advances in co-delivery systems.

Long-term evaluation of functional recovery and nerve regeneration following tubulation repair of nerve defects in mice / 解剖学报

Objective This study is to identify long-term functional recovery and maturity of regenerated nerve fibers after repairing mouse nerve defects with chitosan/polylactide-co-polyglycolide artificial nerve grafts ( CPANGs ) . Methods Mouse sciatic nerve defects, 2mm in length, were bridged by CPANGs (n=6), with nerve autograft (n=6) and nerve defect (n=6) as controls.Plantar test, electrophysiological examination and laser Doppler perfusion imaging following nerve crush were carried out at 1 year after repair to assess nerve function recovery , while muscle wet weight ratio, histological assessment and transmission electron microscopy were performed to evaluate nerve re -innervation and maturity of regenerated nerve fibers .Results When compared to the autograft group , the CPANG group did not show statistically significant difference in functional recovery in terms of paw withdrawal latency , neurogenic vasodilatation , amplitude and latency of compound muscle action potentials ( CMAPs ) , wet weight ratio of gastrocnemius and tibialis cranialis muscles , number of myelinated nerve fibers and density of unmyelinated axons .However , both these two repair groups exhibited significantly longer CMAPs latency , thinner myelin sheath and a lag-behind shift of diameter distribution of myelinated axons as compared to the normal control .Conclusion At 1 year after the mouse sciatic nerve defect was repaired by CPANGs , sensory and autonomic nerve function , number of regenerated axons and muscle re-innervation degree were recovered to the same extent as nerve autografting , but the regenerated nerve fibers were in a state of immaturity .

Polymeric nanoparticle formulation of Octapeptide (NP-OP): In vitro release and in vivo effect in common marmosets, Callithrix jacchus Linn.

yielded an average particle size of 120 nm with 70% encapsulation-efficiency. In vitro release profile of NP-OP showed sustained release of OP for 21 days. In vivo anti-fertility studies were conducted in marmosets. Results indicated that control animals conceived in the same cycle while two of three treated animals failed to conceive in treatment cycle. The in vivo studies thus corroborate with in vitro release of OP, demonstrating its anti-fertility activity in 66% of animals.

Bioabsorbable tension band for the treatment of ulnar styloid fracture / 中国组织工程研究

10.3969/j.issn.2095-4344.2013.25.024

RGD-modified polylactide-co-glycolic acid tissue engineering scaffolds for bone regeneration: An advance / 第二军医大学学报

Polylactide-co-glycolic acid (PLGA) tissue engineering scaffolds play an important role in the regeneration of bone and cartilage. Due to the poor hydrophilicity of PLGA, it is difficult for cells to attach to the scaffolds. Modification by RGD (Arg-Gly-Asp) peptides can effectively improve the cellular affinity of PLGA and adhesion and proliferation of the seed cells. This review summarizes the recent progress in PLGA tissue engineering scaffolds modified by RGD peptides.

Preparation of glial cell line-derived neurotrophic factor loaded microspheres and their in vitro releasing character / 中华创伤杂志

Objective To evaluate the effect of different preparation processes on preparation of the glial cell line-derived neurotrophic factor(GDNF)loaded microspheres and observe the biological activity of GDNF.Methods With polylactide-co-glycolide(PLGA)as the coating material,the GDNF-loaded microspheres were prepared by using double emulsion(W1/O/W2).Two-factor factorial design variance analysis was done to analyze the effects of the composition proportion of lactic acid(LA)and glycolic acid(GA)in PLGA and the stirring speed of multiple emulsion on particle size,entrapment efficiency,burst release and in vitro release characteristics of the GDNF-loaded microspheres.PC-12 bioassay was employed to detect the biological activity of the released GDNF so as to determine the optimal preparation process.Results The composition proportion of PLGA could affect the microspheres'burst release(P ＜ 0.05),with no effect on particle size and entrapment efficiency.with the higher.With higher proportion of GA,the release speed of GDNF in the microspheres was increased.When the stirring speed of multiple emulsion was increased from 1 000 r/min to 3 000 r/min,the particle size of the microspheres was decrease significantly(P ＜ 0.01),the burst release was increased markedly(P ＜ 0.01)and the in vitro release rate was accelerated.The activity of GDNF in the microspheres could last for about 20 days at 37℃,which was 10 days longer than that of single GDNF.Conclusions Double emulsioncan prepare the GDNF-loaded microspheres with high entrapment efficiency and suitable in vitro release time.In the meantime,the microspheres can extend the validity of GDNF.

Development of PEG-PLGA nanoparticles as drug carrier with tumor-targeted therapy / 国际外科学杂志

In recent years, tumor is a refractory disease occurring frequently which is the main cause of death. Surgery, radiotherapy and chemotherapy are the usual therapeutic tools. However,radiotherapy and chemotherapy have serious side-effects and surgery can not be used effectively when metastasis happened. Therefore, tumor-targeted therapy has developed as a better way to cure tumor. Development of research on the use of PEG-PLGA nanoparticles as drug carriers are reviewed in this article, furthermore, problems about that are analysed.

Fixation with Bioabsorbable Polylactide Plate and Screws for the Treatment of the Ankle Fractures / 대한족부족관절학회지

PURPOSE: The purpose of this article is to assess the efficacy of a bioabsorbable polylactide (PLA) plate and screw for treating injuries of ankle fractures. MATERIALS AND METHODS:24 patients who underwent an open reduction and internal fixation operation for ankle fractures from July 2005 to March 2007 were enrolled into the study. There were 15 men and 9 women. The average age of the patients was 44 years and the average follow-up period was 16 years and two months (16.2 months). All cases were divided into low grade fracture patient (11) who belongs in type A and B of Danis-Weber classification and high grade fracture patient (13) who belongs in type C1, C2 of Danis-Weber classification, and each groups were analyzed by clinical (Meyer score) and radiological finding at the time of their last follow-up evaluation. RESULTS:The clinical results according to Meyer scoring system, showed that all patient with low grade fracture had good to excellent result, but only 54% of patient with high grade fracture had good to excellent result. According to Cedell's radiologic finding, there were 91% cases above fair in low grade fracture. But there were 62% of patient above fair result in high grade fracture, the reduction losses were seen in 38% of patient with high grade fracture. CONCLUSION: Bioabsorbable PLA plate and screw is good internal fixation device which doesn't have additional operation for removal of implant because of slow absorption within the human body. It showed sufficient strength for acquisition and maintenance of reduction in low grade fracture, but need attention to use because of many cases of reduction loss in high grade fracture. So, it seems to be safe and effective when used in heeling of low grade fracture under considering about type of fracture sufficiently.

Biocompatibility of new absorbable material:nano-composite of poly (L-lactide) and surface grafted hydroxyapatite / 吉林大学学报(医学版)

Objective To make a comprehensive evaluation of nano-composite of poly(L-lactide) and surface grafted hydroxyapatite(PLLA/PLLA-gHA) as a new material.Methods According to the evaluated critera of medical implanted materials biology and animal trial recommended in GB/T 16886 and IS0 10993 criterion,the new material was carried out on acute systemic toxicity test,haemolysis test,muscular implantation test and subcutaneous injection test.The extract liquid of new material was injected into mice by vena caudalis to test common station,toxic reaction of it at different time,the results were used to evaluate the acute systemic toxicity.Fresh anticoagulant cony blood was mixed with extract liquid of new material with density of 100 g?L-1 to measure each absorbance with spectrophotometer and work out the corresponding rate of haemolysis.The red punctuation and hydropsia of rabbits were observed at different time by subcuntaneous injecting extract liquid into the back of rabbits.PLLA/PLLA-gHA composite plates were implanted into the sacrospinal muscle of rabbits.Cony venous blood was extracted to detect indicatrix of hematology at diferrent time.The material and surrounded tissues were taken out from animals at the 14th,30th,60th,90th,180th,360th day to examine anatomic and pathological changes.Results Rabbits with PLLA/PLLA-gHA composite had good general condition.There was no any acute systemic toxicity in vivo.Data of AST and Scr had no significant difference between experimental group and control group.The hemolysis rate of extrac liquid was 1.22%,which was under the standard criteria(5%).No red punctuation and light hydropsia were observed at different time in the subcutaneous injection test.The inflammation cytochange of PLLA/PLLA-gHA composite group was similar with that of control group in early days,which was met with the general regularity of inflammatory outcome.The fibrosis membrane surrounding the PLLA/PLLA-gHA composite became thinner gradually with the elongation of implantation time.The fibrosis membrane grew into the material at the 360th day.The degree of the fibrosis membrane was below class Ⅰ.Conclusion The new absorbable type PLLA/PLLA-gHA composite has excellent biocompatibility and security.

The Effect of Polylactide Resorbable Film on the Epidural Fibrosis After Lumbar Laminotomy / 대한정형외과연구학회지

PURPOSE: The purpose of this study is to evaluate the effect of polylactide resorbable film in preventing epidural fibrosis after lumbar laminotomy. MATERIALS AND METHODS: Thirty-six New Zealand male rabbits about fifteen month old, weighing 3.5 kg in average, were used. Uniform laminotomy windows measuring 7x7 mm were made on the left side of the spine at three lumber locations (L2, L3 and L4). The laminotomy window at second lumbar spine was left untreated and used as control group (Group 1). The lanimotomy window of 3rd lumbar spine was covered with free fat tissue (Group 2), and polylactide ( Surgi-Wrap(R)) resorbable film was inserted on the laminotomy window of 4th lumbar spine (Group 3). Twelve rabbits were euthanized at 4, 8 and 12 weeks each after operation for biochemical analysis of hydroxyproline content and histologic examination. RESULTS: 1. There was considerable reduction in the total collagen, ranging from 40~48% in the Group 2 or the Group 3 compared to the Group 1. There was no statistical difference between the Group 2 and the Group 3. 2. The extent of fibrosis and fibroblast density was significantly lower in the Group 2 or the Group 3 than in the Group 1. However, the extent of fibrosis and fibroblast density did not differ statistically between the Group 2 and the Group 3. 3. No significant statistical differences of the collagen content and the histologic findings in each group was demonstrated during experimental period. CONCLUSION: These results suggest that polylactide (Surgi-Wrap(R)) resorbable film may be a useful method for reduction of epidural fibrosis after lumbar laminotomy.

Fixation with Bioabsorbable Polylactide Screws for the Treatment of the Ankle Fractures with Syndesmotic Injuries / 대한정형외과학회잡지

PURPOSE: The purpose of this article is to show the efficacy of a bioabsorbable polylactide (PLA) screw for treating syndesmotic injuries of ankle fractures. MATERIALS AND METHODS: Ten patients who underwent an open reduction and internal fixation operation for ankle fractures that had syndesmotic injuries from Dec. 1992 to Feb. 1997 were enrolled into the study. There were 6 men and 4 women. The average age of the patients was 42.6 years and the average follow-up period was 2 years 7 months. The cases were analyzed by clinical and radiological findings at the time of their last follow-up evaluation. RESULTS: According to the clinical results, there was statistically no significant difference between finding an average of 15.8 degrees of dorsiflexion and 45.8 degrees of plantar flexion of the ankle on the affected side of the individuals and that of 17.8 degrees of dorsiflexion and 48.5 degrees of plantar flexion on their normal contralateral side. Upon radiological analysis, the medial clear space was decreased from 7.3+/-1.8 mm to 3.1+/-1.0 mm, the tibio-fibular clear space was decreased from 7.8+/-0.9 mm to 4.9+/-0.5 mm and the tibio-fibular overlap increased from 4.6+/-1.8 mm to 10.5+/-1.4 mm. Also there was no pain and instability in the affected side in any of the patients at the time of the last follow-up. CONCLUSION: A bioabsorbable PLA screw is a good implant for stabilizing syndesmotic injuries of the ankle fractures because there is no evidence of a significant osteolysis resulting around the implant, there is a gradual transfer of stress to the surrounding bone, and there is no requirement for a subsequent removal.

Preparation and release in vitro of carbazochrome hemostasis aerosol film / 第二军医大学学报

Objective: To design a biodegradable aerosol film for treating various wound and diffuse hemorrhage in various organs. Methods: Uniform design was used to screen prescription. Ultraviolet spectrophotometry was used to assay the release in vitro . Results: Time of film formation was less than 30 s. The release in vitro was about 50% in 15 min and stabilized gradually after 15 min. Conclusion:The film formation and release in vitro are well acceptable. [