# Engineering Biomimetic Gelatin Based Nanostructures as Synthetic Substrates for Cell Culture
## 1 Introduction  
The basement membrane (BM) is a specialized form of the extracellular matrix (ECM) present directly under the basal surface of epithelial and endothelial tissues of eumetazoans, where it supports and interacts with other components of the ECM and epithelial cells to promote cell proliferation, migration, and differentiation [1; 2; 3]. Although the BM is known to possess some barrier functionality (to cell and macromolecular migration), it is a relatively porous (pore size \(\sim\)10-130 nm [2]) nanofibrous network mainly composed of a self-polymerizing network of collagen IV and laminin which are connected to one another by glycoproteins, namely nidogen and heparin sulphate proteoglycan [1; 2; 4; 5]. The structure, thickness, and composition of the BM differ with location and tissue type, healthy ageing, and disease [1; 2; 6; 7]. With respect to the latter, examples include asthma and collagenous colitis--a type of nonspecific inflammatory bowel disease which results in an increase of subepithelial BM thickness [8; 9]. In the field of drug delivery, while the drug barrier functions of mucus and epithelium are relatively well-characterized, the filter function of the BM is seldom studied [10; 11; 12]. But currently, there is a growing interest to understand the barrier function of the BM due to advances in the mucosaldelivery of complex therapeutics such as macromolecules and nanomedicines [12; 13]. The diffusion of macromolecules through the BM of non-keratinized oral mucosal epithelium was initially studied by [PERSON] et al., who observed that the BM restricts the penetration of smaller macromolecules (inulin, 5 kDa), while allowing easy passage of larger macromolecules (Dextran, 20 kDa) [14]. This restriction of smaller macromolecules in this study was considered as the result of adhesion or penetration of inulin at the crevices of BM structural proteins [14]. Recently, a similar study was performed by [PERSON] et al. who observed that decellularized BM synthesized by airway epithelial cells restricts the diffusion of macromolecules in a size-dependent manner [12]. There is a need for a well-characterized biomimetic BM substitute which could efficiently replicate the native BM for research in the areas of tissue engineering and drug delivery [15; 16; 17; 18]. Our study focused on the biomimicry of intestinal epithelial BM due to its relevance in drug and nutrient absorption/delivery studies, investigations on intestinal diseases, and infection studies [15; 16; 17; 18]. These studies currently commonly utilize the Caco-2 cell model [14; 15; 17]. This model lacks a proper BM and therefore fails to replicate the complex substrate nanoenvironment taking into account the size, geometry, roughness, stiffness, porosity, and macromolecular composition of the BM [12; 19; 20]. Naturally derived matrices exist, which consist of BM extracts (Matrigel: BM extract secreted by Engelbrecht-Holm-Swarm mouse sarcoma cells) and purified BM proteins (Laminin 111, fibrin gels and hyaluronic acid gels); however, these systems have shown to be unreliable due to their tumor derived nature (which restricts its use in clinical translational studies), lot-to-lot variation, and the inability to understand and decouple their mechanical and biochemical properties [21]. For example, a change in mechanical properties such as matrix density resulted in a change in biochemical properties, such as fiber density and structure, making it impossible to alter one property without disturbing the other [21]. As a result, there is a growing interest and need to fabricate synthetic substrates or BM mimetics with features and properties of human native BMs, as well as easily tunable bioactivity and mechanical properties [12; 21]. Electrospinning is a well-known versatile, cost-effective, and commonly used nanofabrication method which can be easily upgraded for industrial-scale production of nanofibers [22].