Study On Hyaluronan Micro/Nano-spheres As Drug Delivery System

Micro/Nano-spheres as drug release system made of natural polymers have played many competent roles in controlled release of drugs. Among them, hyaluronan (HA) is a kind of consistent glycosaminoglycans in extracellular matrix, and has wonderful biocompatibility, which is promising to be developed as drug carriers to realize controlled release. However, natural HA is water-soluble and easy to be absorbed in tissues, and thus is limited to be applied to prepare drug carriers asking for better rigidity, mechanical intensity and stability.Chemical cross-linking is an effective method to enlarge the molecular weight of HA, prolong its time of degradation and improve its stability. The purpose of this project is mainly to discuss the rule of cross-linking HA in anhydrous system, develop methods to prepare HA micro/nano-spheres as drug carriers, study their properties and the drug release behavior, and prove the possibility of applying HA magnetic micro/nano-spheres in the field of magnetic drug delivery in the near future.(1) Cross-linking of HAWe use seven cross-linkers: adipic dihydrazide (ADH), glutaraldehyde (GTA), Trisodium trimetaphosphate (STMP), divinyl sulfone (DVS), N-N' dicyclohexyl carbodimide (EDC), ethylene glycol diglycidyl ether (EX-810) and genipin (GP), to cross-link HA in multi-phase system by applying dehydrated alcohol as reaction medium. We discuss the influence of time of reaction, pH of reaction medium and reaction temperature to the procedure of cross-linking. After rinsing, the products are swelled in 50% alcohol-PBS. Based on the phenomenon of swelling and kinatic viscosity (η) of the steeps, we assess the products prepared by each cross-linker, and make sure the best conditions of reaction.FT-IR and ¹H-NMR indicate that ADH and EDC cross-link HA by forming carboxyl, whereas the other five cross-linkers by forming ether bond. T₁ of ¹H-NMR verify the cross-linking effect. By testifying HA derivative, T₁ shows higher molecular weight of HA-GTA, HA-STMP and HA-EDC than pure HA.By analyseη, the significant of cross-linking effect is HA-GTA＞HA-EDC＞HA-STMP＞HA-ADH＞HA-GP＞HA-EX-810＞HA-DVS＞HA, and generally, in anhydrous system, cross-linking can improve molecular weight of HA to form sole instead of swelled linear polymer in procedure of swelling, and strengthen its capability to prolong degradation procedure in enzyme solution, but the acidic or alkaline condition of reactant also degrades HA in the reaction. The capability of HA cross-linking derivatives to confront degradation by HAse is: HA-ADH＞HA-STMP≈HA-GTA＞HA-EDC＞HA-GP＞HA-DVS＞HA-EX-810。Cellular toxicity, hemolysis and Intracutaneous stimulation tests illustrate that HA-STMP is the only HA derivatives that past all the tests, and above all, STMP is the most satisfying cross-liker.(2) Forming HA-STMP cross-linking microspheresUsing technique of ultrosound and inversed phase (W/O) emulsification, HA-NaCl solution as water phase, n-heptane as oil phase, SPAN and TWEEN as emulsifier, STMP as cross-linker, NaOH solution as regulator of pH, we form HA-STMP microspheres with wonderful shape and property of dispersion (observed by optical microscope), which can constrict to nanospheres after dried (observed by SEM).An orthogonal experimental design and statistical analysis method are employed to optimize the condition of experiment. Mean diameter (d) and polydispersity index (PDI) as the variables, different criterions to assess experiment is built, and the optimized condition is given with the sequence of significance concerning to the influence the factors to d and PDI:1) d to minimum, PDI to minimum: STMP/HA (structural unit, similar to below) =1/2(M/M) )>(HA)=0.2%>Emulsifier/Oil=1/100(g/g)>Water/Oil=1/4(V/V).2) d to maximum, PDI to maximum: STMP/HA=I/50(M/M)>C_HA=0.5%>E/O=1/80(g/g)>W/O=1/4(V/V).3) d to minimum, PDI to maximum: E/O=1/40(g/g)>STMP/HA=1/5(M/M)>C_HA=1%>W/O=1/4(V/V).4) d to maximum, PDI to minimum: E/O=1/100(g/g) >STMP/HA=1/10(M/M)>C_HA=0.2%>W/O=1/3(V/V).(3) Forming dexamethasone-HA-STMP cross-linking microspheres (DEX-HA MS) and ligustrazine-HA-STMP cross-linking microspheres (LIZ-HA MS)Based on the optimization designs above, we add drug into water phase, and use W/O ultrasound emulsification to form microspheres as drug carriers. The shape of drug carriers has little change from the micro/nanospheres, but d is a little larger. As to LIZ-HA MS, d of LIZ-HA is larger, but d of all the micro/nano-spheres are within the same order of magnitude. There is a negative correlation between d and the amount of STMP, between d and the amount of emulsifier, and positive correlation between d and C_HA.We test drug loading% (DL%), loading efficiency% (LE%), and cumulative release%(CR%) of in vitro drug release experiment. When C_HA≦1%, drug/HA≦1/10 (g/g), there is a positive correlation between the factor mentioned below and DL%/LE%: the amount of STMP, the amount of emulsifier, the amount of HA and the amount of drug. DL% and LE% of LIZ -HA MS is generally lower than those of DEX-HA MS. As to CR% of DEX-HA MS and LIZ-HA MS, the degree fits in with pervasion-corrosion equation, and is higher than that with Higuchi equation. There is a negative correlation between the factor mentioned below and CR%: the amount of STMP, the amount of emulsifier, and C_HA. When DEX/HA≦1/5(g/g) or LIZ/HA≦1/15(g/g), there is a negative correlation between the amount of drug and CR%. CR% of LIZ-HA MS is higher than that of DEX-HA, under the premise of using the same formula for preparation.(4) Forming rapamycin-HA microspheres (multiple emulsion)We improve salt out method to form rapamycin-HA microspheres (RPM-HA-MS), Using RPM-HA single phase as continuous phase, dehydrated alcohol-NaCl saturated solution as dispersed phase. Under×1000 oil microspheres, we have observed that the structure of the RPM-HA MS has clear multi emulsifier.We change the components of saturated solution, find out that dehydrated is helpful to deposit microspheres, but n-heptane has little function about these.When continuous phase/dispersed phase=1∶3 (V/V) and C_{RPM-HA(single emulsion)}≦0.5%, these two factor has positive correlation with DL%, and influence little about CR%. There is a positive correlation between the amount of RPM and DL%. In vitro drug release experiments shows the release rate of RPM-HA MS is faster than DEX-HA MS.We apply a RPM-HA MS to intromascular injection experiment on rabbits, RPM standard as control. HPLC tests the concentration of drug release procedure. The result shows RPM-HA MS is a slow-regarding drug release system.(5) Forming DEX-HA magnetic microspheresMagnetic fluid is prepared based on relative reports. We add magnetic fluid into water phase consist of HA-NaCl and drug, and stir water phase to be well-proportioned, W/O ultrosound emulsification to prepare HA magnetic microspheres as drug carrier. The products show wonderful shape and dispersion, and magnetic response experiment demonstrates the superparamagnetism of HA magnetic microspheres. There is little difference about d, DL%, LE% between magnetic microspheres and non-magnetic microspheres. So, HA magnetic microspheres has optimistic prospect in the field of target-oriented drug delivery systems.