Study On The OA Therapy Using PAMAM-KGN Drug Delivery System Based On MSCs And Its Molecular Pharmaceutical Mechanism

Osteoarthritis?OA?is a common degenerative disease of the articular joints,which affects millions of people around the world.Intra-articular?IA?injection of pharmacologic agents is an effective measure for alleviating the symptoms and preventing the progression of OA.However,it is difficult for efficient delivery of agents due to the rapid clearance of small molecular or biomacromolecules from the joint cavity and the dense cartilage extracellular matrix?ECM?.These are limitations to the development of anti-OA medications.So,the study intends to construct a new intra-articular?IA?drug delivery system for the treatment of osteoarthritis,especially for the repair of articular cartilage injury and cartilage targeting and retention.This paper is divided into two parts.The first part is to design a“Nano in Gel”composite system,which is a PAMAM-KGN nanoparticle?NPs?loaded thermo-sensitive gel delivery system for the repair of cartilage injury in vivo.Firstly,polyamidoamine?PAMAM?dendrimer was used as a nanocarrier for the cytoplasmic delivery of kartogenin?KGN?.KGN is a small molecule that promotes the selective differentiation of mesenchymal stem cells?MSCs?into chondrocytes.Then we synthesized and compared three different PAMAM-KGN nanoparticles based on the hypothesis that drug binding method may affect the chondrogenic differentiation efficacy.Finally,the optimized PAMAM-KGN nanoparticles were combined with the thermo-sensitive hydrogel to construct a sustained-release composite system.IA injection of the composite system could recruit the bone marrow and synovial-derived mesenchymal stem cells on the hydrogel scaffold and delivery of PAMAM-KGN NPs for cartilage regeneration.The second part is to construct a cartilage targeting delivery system based on the specificity of the cartilage structure,that is,cartilage extracellular matrix?ECM?is a negatively charged 3D meshwork enriched in hyaluronic acid?HA?with a pore size of?60nm.In this part,we developed an efficient intra-articular delivery nanosystem based on polyethylene glycol?PEG?modified PAMAM dendrimer,and a chondrocyte affinity peptide?CAP?was applied to further enhance cartilage selectivity.The nanocarri er could be a useful nanoplatform for intra-articular delivery of therapeutic agents to cartilage.Section 1:PAMAM-KGN NPs incorporated thermogel supports stem cells for cartilage regenerationThe small molecule KGN was synthesized by one step method using phthalic anhydride and 4-aminobiphenyl as starting materials.The structure of KGN was confirmed by ¹H NMR,¹³C NMR,FTIR and MS.As well as the dissolution characteristics and partition coefficient of KGN were measured.The results showed that KGN could be identified as low solubility and high permeability.With KGN and PEGylated PAMAM dendrimer as the starting materials,PAMAM-KGN NPs with three different drug binding methods were prepared,namely physical encapsulation PP/K,covalent attachments PPK and KPP.The conjugated number of PEG per PAMAM dendrimer was similar and calculated to be 20.The average diameter of NPs was around 30-40 nm,spherical morphology,and the Zeta potential was about+5 mV.The drug loading of KGN was in the range of 5.2-5.5%by weight.The bone marrow MSCs?BMSCs?were obtained by the method of whole bone marrow adherence.MTT assay showed that PAMAM-KGN NPs did not affect the normal growth of BMSCs.The uptake of PEG-PAMAM by RAW264.7 macrophages was evaluated by flow cytometry and the results showed that PEGylation modification could protect nanocarrier from RAW264.7 cell phagocytose.The result of in vitro chondrogenic differentiation showed that all of KGN and PAMAM-KGN groups could up-regulate the expression of chondrogenic markers genes compared with no treatment group.The KPP group exhibited the highest expression of chondrogenic markers in all the groups.The uptake,uptake mechanism and intracellular distribution of nanocarriers were investigated by flow cytometry and confocal microscopy.The results showed that the uptake rate of PEG-PAMAM was a fastening process on BMSCs.The nanocarriers were internalized by caveolin-medizted endocytosis,and then only part of them was delivered to the lysosomes.Due to the proton sponge effect,which facilitated the escape of nanocarrier from the lysosomes,the escaped conjugates?PAMAM-KGN?could diffuse into the cytoplasm to competitively interacted with intracellular signal molecules and promote the differentiation of MSCs into chondrocytes.Among them,KPP had the strongest competitive ability.The PLGA-PEG-PLGA block copolymers were synthesized by ring-opening polymerization.The structure was confirmed by ¹H NMR,FTIR and GPC.The physical and chemical properties of the copolymer solution were investigated and then a KPP-loaded thermogel composite system?“Nano in Gel”?was prepared.The results showed that the phase transition temperature of the complex?20 wt.%?was 29 ^oC and the fluidity at room temperature is good,so could be used for intra-articular injection.In vitro release experiments showed that the release time of KGN and KPP NPs from thermogel was up to 27 days,which played a sustained release effect on the drug.KPP NPs incorporated thermogel supports stem cells for cartilage regeneration in vivo was studied.The biocompatibility of the KPP-loaded thermogel complexes was evaluated by in vitro BMSCs cell proliferation assay,subcutaneous and intra-articular injection of complexes for pathological examination.The results showed that the biocompatibility of KGN/Gel and KPP/Gel was good.The results of in vivo imaging showed that the nano-drug system combined with the thermo-sensitive gel prolonged the residence time of the drug in the OA rat joint cavity,thus contributing to enhance the effect of KGN.The OA model of rat was induced by intra-articular injection of pepain.Intra-articular injection of KGN delivery system could induce MSCs into chondrocytes to repair damaged cartilage.Gross morphological score and histological scores of articular cartilage revealed that the repair tissue in the group of KPP loaded-thermogel was close to the natural hyaline cartilage.Section 2:Chondrocyte affinity peptide modified polyamidoamine dendrimer as a nanoplatform for targeting and retention in articular cartilageFirstly,flow cytometry was applied to screen cartilage affinity peptides?CAP?with high uptake efficiency on chondrocytes.Then rhodamine B isothiocyanate?RBITC?was bonded to the surface of PAMAM to obtain PAMAM-RB for intracellular tracing and biological evaluation.Finally,we conjugated CAP to PAMAM-RB by the dual-function Mal-PEG-NHS to prepare a series of CAP-PP-RB conjugates with different CAP number,namely CAP4-PP-RB,CAP8-PP-RB and CAP16-PP-RB.The conjugated number of CAP peptide per PAMAM was calculated to be 3.7,6.6 and 12.4,respectively.The CAP-PP-RB conjugates have a particle size range of 50-60 nm and a Zeta potential of+4.5-+5.5 mV.The results of flow cytometry revealed that the cell uptake of CAP-PP-RB decreased with increasing CAP conjugated number.CAP4-PP-RB could be used to evaluate the effects of CAP modification on the in vitro and in vivo assays.The uptake,uptake mechanism,intracellular distribution and penetrating ability of CAP4-PP-RB and PP-RB were investigated by flow cytometry and confocal microscopy.CAP4-PP-RB exhibited much more selective and efficient cellular uptake by chondrocytes than that of PP-RB.Uptake kinetics of conjugates presented a dose-and time-dependent behavior of cellular uptake.The cellular uptake mechanism and intracellular localization study revealed that both of conjugates were likely internalized by chondrocytes via clathrin and caveolin co-mediated endocytosis,and delivered to lysosomes.Importantly,the CAP4-PP-RB showed enhanced cartilage-targeting and penetration effect in vitro 3D multicellular chondrocytes spheroids.In vivo imaging demonstrated the fluorescein Cy7 labeled CAP4-PEG-PAMAM was capable to persist in the joint cavity of rats for a prolonged time when compared with free Cy7.The biocompatibility of the nanocarrier in the joint was good and did not cause obvious cartilage injury and synovial inflammation;In vivo cartilage targeting and penetration experiments showed CAP4-PEG-PAMAM could effectively penetrate into cartilage tissue and specifically deliver the therapeutic agent into chondrocytes.