Construction, Characterization & Application Of Thermosensitive Hydrogel As An Embedding Carrier

1. Construction of thermosensitive hydrogel as an embedding carrierBACKGROUND: Thermosensitive hydrogel Poly-(N-isopropylacrylamide) (P(NIPAAm)) and its ramifications are widely used in drug delivery, cell culture and tissue engineering, but have not been evaluated as medical implant for clinical use. OBJECTIVE: To construct a thermosensitive hydrogel and regulate it LCST for proper use as an embedding carrier.DESIGN:In vitro study UNIT:Orthopaedics department of Union Hospital, Tongji Medical College, Huazhong University of Science & TechnologyMATERIALS:NIPAAm monomer and NHMPA monomer from Aldrich Co., crosslinking agent N, N'-methylene bisacrylamide (MBAAm) from Fluka Co., initiator ammonium persulfate (APS) and accelerating agent tetramethyl ethylene diamine (TEMED) from Sigma Co., other reagent were analytical pure.METHODS: We used APS and TEMED as an oxidoreduction initiation system and MBAAm as cross-linking agent to implement the polymerization, some amount of NHMPA was added into reaction system to regulate LCST which was determined by shrinking tests. MAIN OUTCOME MEASURES:Correlation analysis was made between the LCST and the amount of NHMPA used to yield a linear relationship.RESULTS:Synthesized Hydrogel showed thermosensitive character as expected and the LCST was 38.5℃while some amount of NHMPA were added into the reaction system. CONCLUSION:The thermosensitive hydrogel P(NIPAAm-co-NHMPA) has a proper LCST of 38.5℃and might become a good medical implant for clinical use.2. Improving thermosensitive hydrogel by adding several chemical componentsBACKGROUND: Thermosensitive hydrogel Poly-(N-isopropylacrylamide) (P(NIPAAm)) and its ramifications are widely used in drug delivery, cell culture and tissue engineering, but have not been evaluated as medical implant for clinical use. OBJECTIVE: To improve the thermosensitive hydrogel for faster thermo-responsibility and better mechanical function by adding several chemical components for proper use as an embedding carrier.DESIGN:In vitro studyUNIT:Orthopaedics department of Union Hospital, Tongji Medical College, Huazhong University of Science & TechnologyMATERIALS:NIPAAm monomer and NHMPA monomer from Aldrich Co., crosslinking agent N, N'-methylene bisacrylamide (MBAAm) from Fluka Co., initiator ammonium persulfate (APS) and accelerating agent tetramethyl ethylene diamine (TEMED) from Sigma Co., NaHCO3 and inorganic clay were analytical pure.METHODS:We used APS and TEMED as an oxidoreduction initiation system and MBAAm as cross-linking agent to implement the polymerization, some amount of NHMPA was added into reaction system as the experiment in part I to make sure a LCST proper for implantation, then some amount of NaHCO3 and inorganic clay was added into reaction system separately to evaluate the improvement of thermo-responsibility by NaHCO3 and amendment of mechanical function by inorganic clay. Finally, NHMPA, NaHCO3 and inorganic clay were added together to evaluate the best prescription.MAIN OUTCOME MEASURES:Thermo-responsibility and mechanical function was recorded to decide the best prescription of thermosensitive hydrogel as a medical embedding carrier.RESULTS:After NaHCO3 added, the hydrogel got a better thermo-responsibility than original hydrogel but a poor mechanical function; after inorganic clay added into the reaction system, the mechanical function was much better and finally proper for clinical use.CONCLUSION : The hydrogel we finally obtained has both good function of thermo-responsibility and mechanical function, and its LCST remains above normal body temperature, and that enable its further use in clinical medicine.3. Slow-release function of two thermosensitive hydrogel BACKGROUND: Thermosensitive hydrogel Poly-(N-isopropylacrylamide) (P(NIPAAm)) and its ramifications are widely used in drug delivery, cell culture and tissue engineering, but have not been evaluated as medical implant for clinical use.OBJECTIVE: To evaluate the slow release function of the thermosensitive hydrogel so as to decide its carrier ability.DESIGN:In vitro comparison studyUNIT:Orthopaedics department of Union Hospital, Tongji Medical College, Huazhong University of Science & TechnologyMATERIALS:NIPAAm monomer and NHMPA monomer from Aldrich Co., crosslinking agent N, N'-methylene bisacrylamide (MBAAm) from Fluka Co., initiator ammonium persulfate (APS) and accelerating agent tetramethyl ethylene diamine (TEMED) from Sigma Co., NaHCO3, inorganic clay , other reagents were analytical pure.METHODS:The in vitro release experiments were carried out at 37℃and 42℃respectively to investigate the effect of temperaturesensitive property of the hydrogel on adriamycin release profiles. Release experiments were conducted by immersing the swollen adriamycin loaded hydrogel in a glass tube, filled with a 25mL PBS (0.1M, pH 7.4) at 37℃(below LCST) or 42℃(above LCST) with a shaking rate of 80 rpm. At a predetermined period of in vitro release experiment, 2mL aliquots of the buffer medium was removed from the glass tube and the concentration of adriamycin in that aliquot was measured by using a UV spectrophotometer at 475 nm while 2 mL fresh buffer solution was added back to the glass tube to maintain the same total solution volume. For the comparison purpose, hydrogel without Clay and NaHCO3 was used as a control. Then we established a tissue modeling system by agrarose to describe the in vivo release curve. Adriamycin loaded hydrogel was put in center of agrarose, and the adriamycin released was measured and recorded by spectrophotometer to draw a release curve in vivo.MAIN OUTCOME MEASURES: The concentration of adriamycin in that aliquot was measured by UV spectrophotometer (475 nm) to study the slow release function in vitro of thermosensitive hydrogel under different temperature and the in vivo release function was modeled by agarose modeling system.RESULTS:The hydrogel could slow release adriamycin, and the release velocity showed temperature sensitivety. When surrounding temperature rised LCST, the release velocity of hydrogel splendid jumped to a much higher platform at the same time.CONCLUSION:P(NIPAAm-co-NHMPA) has good slow release function and its release function was temperature responsiveness, that character enabled the hydrogel to become a good medical implant for clinical use. persulfate (APS) and accelerating agent tetramethyl ethylene diamine (TEMED) from Sigma Co., NaHCO3, inorganic clay , other reagents were analytical pure.METHODS:The in vitro release experiments were carried out at 37℃and 42℃respectively to investigate the effect of temperaturesensitive property of the hydrogel on adriamycin release profiles. Release experiments were conducted by immersing the swollen adriamycin loaded hydrogel in a glass tube, filled with a 25mL PBS (0.1M, pH 7.4) at 37℃(below LCST) or 42℃(above LCST) with a shaking rate of 80 rpm. At a predetermined period of in vitro release experiment, 2mL aliquots of the buffer medium was removed from the glass tube and the concentration of adriamycin in that aliquot was measured by using a UV spectrophotometer at 475 nm while 2 mL fresh buffer solution was added back to the glass tube to maintain the same total solution volume. For the comparison purpose, hydrogel without Clay and NaHCO3 was used as a control. Then we established a tissue modeling system by agrarose to describe the in vivo release curve. Adriamycin loaded hydrogel was put in center of agrarose, and the adriamycin released was measured and recorded by spectrophotometer to draw a release curve in vivo.MAIN OUTCOME MEASURES: The concentration of adriamycin in that aliquot was measured by UV spectrophotometer (475 nm) to study the slow release function in vitro of thermosensitive hydrogel under different temperature and the in vivo release function was modeled by agarose modeling system.RESULTS:The hydrogel could slow release adriamycin, and the release velocity showed temperature sensitivety. When surrounding temperature rised LCST, the release velocity of hydrogel splendid jumped to a much higher platform at the same time.CONCLUSION:P(NIPAAm-co-NHMPA) has good slow release function and its release function was temperature responsiveness, that character enabled the hydrogel to become a good medical implant for clinical use. MAIN OUTCOME MEASURES:L929 cells were maintained and the cytotoxicity test was carried out by normal MTT method; the cells/scaffolds interaction test was detected by microscopic; the hemolysis test was meatured by hemolysis rate; pyrogen test was meatured by bacterial endotoxin detection. The erythema and edema of bilateral spine of guinea pigs were recorded in sensitization test; the general state of each mouse in acute systemic toxicity test were recorded 4, 24, 48 and 72h after injection; all mice in genetic toxicity test were executed six hours after injection and marrow polychromatic erythrocyte(PCE)micronucleus was counted under microscope; 1, 4 and 8 weeks after implantation, two rabbits were executed respectively and the muscle about 0.5cm around the material was cut, made into sections and observed under light microscope.RESULTS:In cytotoxicity test, the toxicity rate was 0-1. Through the scanning electron microscopy, cells and scaffolds were tightly joined, producing a perfect interface, and the shape of cells was all right. Hemolysis rate was 2.54%. No pyrogen reaction. In sensitization test, there was no erythema and edema occuranced after leaching liquor and saline injected while positive result was seen after formaldehyde injected; in acute systemic toxicity test, the symptom of toxicity was not found in neither experimental group nor control group but formaldehyde injection group showed positive result; the genetic toxicity test suggested no difference between experimental group and negative control group while control group with cyclophosphamide injection showed positive result; the implantation test showed that the inflammation around the material was mild.CONCLUSION:P(NIPAAm-co-NHMPA) has good biocompatibility in vitro/vivo and might become a good medical implant for clinical use.5. Therapeutical effect of thermosensitive adriamycin slow-release hydrogel against mouse osteosarcoma in vivo BACKGROUND: Thermosensitive hydrogel Poly-(N-isopropylacrylamide) (P(NIPAAm)) and its ramifications are widely used in drug delivery, cell culture and tissue engineering, but have not been evaluated as medical implant for clinical use.OBJECTIVE: To study the therapeutical effect of a novel thermosensitive adriamycin slow-release hydrogel P(NIPAAm-co-NHMPA) against mouse osteosarcoma in vivo.DESIGN:Random, no-blind, group control, animal experimental study UNIT:Orthopaedics department of Union Hospital, Tongji Medical College, Huazhong University of Science & TechnologyMATERIALS:NIPAAm monomer and NHMPA monomer from Aldrich Co., crosslinking agent N, N'-methylene bisacrylamide (MBAAm) from Fluka Co., initiator ammonium persulfate (APS) and accelerating agent tetramethyl ethylene diamine (TEMED) from Sigma Co., other reagent were analytical pure.METHODS:The tumor bearing mice were divided into two groups: drug containting hydrogel was embedded into experimental group and empty hydrogel was embedded into control group.MAIN OUTCOME MEASURES:General state of animal health was observed and tumor growth state was recorded to draw a tumor growth curve. At the end of observation, frozen sections were made for microscope observation. RESULTS:Imbedding drug containing hydrogel could inhibit the growth of mouse osteosarcoma in vivo; at the contact surface between drug containing hydrogel and tumor tissue, cell necrosis was found under microscope.CONCLUSION:P(NIPAAm-co-NHMPA) could slow release adriamycin and inhibit the growth of mouse tumor in vivo, that might become a good medical implant for clinical use.