In vitro and ex vivo characterization of nanonized amniotic membrane particles: An untapped modality for ocular surface reconstruction

The pristine Human Amniotic Membrane (HAM) has portrayed outstanding potential as scaffold for ocular surface reconstruction and regeneration. However, in treatment procedures where the supporting membrane matrix of HAM is not obligatory and only the bioactive molecules are vital, the surgical practise of HAM grafting causes redundant trauma and economic burden to the patient. Hence, in our laboratory we have attempted to break down HAM to nanoscale particles and validate its potential as a competent ocular therapeutic agent; by conducting a comparative analysis between the fresh, lyophilized, micronized and Nanonized Amniotic Membrane (NAM) particles. Our results evidently showcased that the prepared NAM particles was <100 nm and the major biomolecules such as collagen and hyaluronic acid were well retained. Further, the NAM particles eluted significantly higher amounts of proteins and growth factors while maintaining its stability and isotonicity when stored at 4 degrees C. Its biostability was assayed in the presence of lysozyme enzyme. Its remarkable ability to promote cell proliferation in rabbit corneal cells and negative cytotoxicity is an added advantage for ocular application. The ocular biocompatibility of NAM, evaluated by the ex vivo assessment of corneal thickness, transparency, histopathology, immunohistochemistry and corneal permeability clearly indicated its suitability for ophthalmic applications.