Peripheral Arterial Disease (PAD) is a severe syndrome in which arterial blood flow of the lower extremity is reduced or interrupted by various factors such as embolism or atherosclerosiss [4,8,20]. PAD is the major cause of lower extremity amputation with high disability rate, in severe cases it can even cause the death of the patients [2,31]. Currently, the primary treatments for PAD are arterial thrombectomy and arterial reconstruction which include open surgery and interventional therapy [18,19,24]. However, the current cannot completely solve arterial stenosisin a single treatment cycle [23,27]. PAD patients after receiving surgery often need to continue to take oral medications, such as vascular dilators, antiplatelet drugs and anticoagulants [10,12,35]. Among these drugs, the vasodilator drugs are relatively cheap and basically include prostaglandin analogs, such as cyclic prostaglandins [5,9]. However, their half-life period in serum is extremely short, ranging from a few seconds to tens of minutes, which results in low durability and limited efficacy [5,6,33]. Therefore, durable medicines that extend the half-life period are needed.

Nowadays, gene therapy is becoming one of the most important strategies for PAD besides surgery and medical therapy [14,16,28,34]. By means of virus or non-virus transfection, gene therapy could transport plasmid or recombinant protein to the inside of the cells, which can extend the treatment time and provide a long-term curative effect [22,32]. At present, the commonly used transfection reagents in animal experiments include adenovirus, liposome, polyethyleneimine 25 kDa (PEI25kDa). Compared to liposomes with relatively simple structures and fewer functional groups, the polymer nanoplatform has multiple building blocks, which allow for easier modification of functional groups and flexible applications. Moreover, they can achieve carrier degradation or specific functions in vivo. Traditionally, PEI25kDa is used as the “golden standard” of the cationic polymer with promising transfection efficiency, but it faces high cytotoxicity with its large molecular weight [26]. As a modified cationic polymer gene delivery system, GAPEI is easy to fabricate and can maintain high transfection efficiency while reducing cytotoxicity by modifying small-molecular-weight PEI 1.8 kDa. The polycations can bind with the plasmid through electrostatic force and form different nanostructures, which can facilitate the escape of the nucleic acids through the proton sponge effect and release them to the cytoplasm [11]. But the treatment efficacy of these polycations depends on the complexity of the molecular chains [29]. Hence, ND-PEI800 compensates the shortcomings of PEI25kDa to a certain extent, but the synthesis of ND-PEI800 is time-consuming and subjected to rigorous conditions, which restrict its wide use in PAD drug delivery [36]. However, in general the larger molecular weight polycations have higher transfection efficiency, but the cytotoxicity of the polycations tends to increase. Thus, the development of low cytotoxicity and long-lasting medicines for gene therapy of PAD remains challenging.

In this work, glutaraldehyde (GA) was chosen as the cross-linking molecule of PEI1.8 kDa (GAPEI) to form the composites as transfection reagents coating prostacyclin synthase (PTGIS) plasmid for gene therapy of PAD. Studies have revealed PTGIS was related to PAD therapy. Meegan et al. have found that PAD patients suffered from blood flow impairment, which was correlated with the amount of PTGIS [33]. Under pathological conditions, the expression of Cyclo-oxygenase (COX)-2 isoform was increased, and it could be part of an ‘anti-arteriosclerotic’ defense mechanism through its main metabolite, prostacyclin [3,21,25]. Thus, PTGIS plasmid is used in PAD therapy to inhibit atherosclerosis and replace traditional vasodilators. As a low molecular weight polymer, PEI1.8 kDa could provide higher transfection efficacy when coupled with glutaraldehyde to form a high molecular weight PEI polymer. In addition, due to the biodegradability of C=N bond formed by the aldehyde groups of GA and amino groups of PEI 1.8 kDa in a long period, the materials showed low cytotoxicity with a high transfection efficacy, even better than that of commercial PEI 25 kDa. This study provides a new concept and method for designing PEI for gene treatment of PAD by realizing both long-term therapeutic effect and low cytotoxicity.