CPP-mediated Ocular Gene Delivery System

Intraocular neovascularization diseases (IND) are ocular disorders of high incidence, including age-related macular degeneration, diabetic macular edema and other occoecatio diseases. Vascular endothelial growth factor (VEGF) plays an important role in IND pathogenesis and has been regarded as a main therapeutic target. Currently, integrin, monoclonal antibody, aptamer and siRNA are used for antagonism of VEGF and neovascularization inhibition in clinic. These biomacromolecules are difficult to penetrate into the eyeball by non-invasive pathway because of their molecular weight and strong hydrophilcity. Intravitreal injection is the current administration for these drugs with a rather low patient compliance. Thus the improvement of non-invasive ocular drug delivery is the focus of pharmaceutical research.Cell-penetrating peptides (CPPs) are positively charged short peptides within 30 amino acid residues with strong cell penetration ability. CPPs get into cells by a receptor dependent way and can effect without cellular damage. Most researched CPPs including TAT, Polyarginines, Penetratin and Transportan have successfully delivered proteins, nucleotides, nanoparticles, and liposomes into cells.This paper aimed at screening an optimized CPP with outstanding ocular permeability and safety from several well-developed CPP candidates and preparing a CPP-mediated non-invasive ocular gene delivery system to promote the posterior segment absorption of gene drugs via eye drop in conjunctival sac. We hope this new system can improve the patient appliance by replaceing the original intravitreal injection method and realize the novel bioactive macromolecules delivery in the eye.The first part accomplished the multiple evaluations of Penetratin, Protamine, TAT, Poly(arginine)g and neutral Poly(serine)g in different levels. By NHC uptake, MTT assay, ex vivo permeability experiments with rabbit cornea and in vivo distribution in rat eyes, we did a systemic research on CPPs ocular permeability. Seen from the results, among these four CPP candidates, Penetratin obtain a strongest ocular permeability and safety. The cell uptake was promoted by 16～29 folds and the ex vivo corneal apparent permeability coefficient by 87.5 folds. It can be absorbed by the eyeball and make a quick and wide distribution in posterior segment after eye drop in conjunctival sac. Through circular dichroism test, we make a comparison of the spatial characters for the four candidates. It seems that the strong ocular permeability of Penetratin comes from its amphipathicity and unique spatial structure called PPII Helix. In addition, we synthesized the D-form CPPs with the same amino acid sequence with the original candidates and administrated a comparative study. It was found that the ocular permeability of CPPs was weakened after interconversion of conformation. As a result, we choose Penetratin in L-form as optimized CPP molecule in the following research.The second part design and prepare two physical complexes in different structures with pRFP, Low molecular weight Branched Cationic Polymer (LBCP) and Penetratin (P):pRFP/P and pRFP/LBCP/P. By packaging the reporter gene by electrostatic bonding, we did the formula optimization, structure verification and physical/chemical characterization. The optimized formula is as follows:charge ratio of Penetratin and pRFP is 20:1 in pRFP/P complex; N/P ratio of LBCP and pRFP is 5:1 and charge ratio of Penetratin and pRFP 20:1 in pRFP/LBCP/P. We then finished the verification of physical complexes by ultrafiltration centrifugation. The particle size of pRFP/P is around 400 nm, pRFP/LBCP/P around 100-200 nm, among which PAMAM participated complexes are much smaller than others informing a stronger gene packaging and Penetratin binding ability. The TEM results prove that the appearances of these physical complexes are round and uniform accordance to the particle size test.The third part made a further evaluation of the formula we got above in both in vitro and in vivo test. With NHC and SDHCEC in vitro models, we did both qualitative and quantitative cellular transfection effeciancy test and research on cell uptake mechanism. The best formulas for ocular gene delivery after screening are pRFP/P20 and pRFP/PAMAM/P20+PAMAM5. The in vivo test on rats proved that the two formulas above could realize a distinguished transfection effect. The ocular pharmacokinetic test showed that both the two complexes could get into the eye and make a long residence more than 8h.In conclusion, the cell-penetrating peptide Penetratin with excellent ocular permeability and biosecurity showed itself through in vitro and in vivo screening design. Gene delivery systems are successfully established utilizing penetratin, pRFP and LBCP. These two achieved gene delivery systems are able to rapidly reach posterior eye segment and properly transfect passing by a non-inasive pathway, which delighted gene therapy for intraocular neovascular diseases.