American Lung Association has revealed the incidence and prevalence of almost 50,000 new cases of Idiopathic pulmonary fibrosis (IPF) every year among the people between 50 and 70 years old [1]. Along the same line, Pulmonary Fibrosis Foundation has provided the statistics of IPF with the active cases of 250,000 patients, new cases of 50,000 patients annually, and a mortality rate of 40,000 people only in the US [2]. IPF is considered as a severe form of idiopathic interstitial pneumonia and contributes to 25% of the cases of interstitial lung disease. The pathology of fibrosis starts due to deregulated wound healing processes in response to any chronic tissue injury or tissue inflammation, and is related to the remodeling of lung parenchyma, excessive deposition of extracellular matrix components and connective tissue components, chronic inflammation, and irreversible lung scarring [3]. The progressiveness of IPF is responsible for distortion in the lung's architecture and leads to the stage of organ failure, thereby impacting the life of patient in terms of morbidity and mortality [4,5]. Historical data revealed the high mortality rate in case of IPF patients, with a median survival of 2–3 years after the diagnosis [6]. Clinical management of IPF patients is a challenging task as the therapeutic options are still limited to treat IPF patients. Despite receiving anti-fibrotic therapy, many patients progress to the end-stage of pulmonary fibrosis, where lung-replacement is the only option for them to get rid of IPF and live a normal life [6,7].

Lately, nindetanib and pirfenidone had been approved by FDA for the treatment of IPF and were found to be effective in treating patients with mild to moderate IPF. However, modified doses were administered to the patients who experienced adverse effects with higher doses [8]. Though these two drugs are available in clinic and prescribed by the clinicians, still promising formulations are required to be developed to overcome their drawbacks and fulfil the unmet needs for the treatment of patients with mild, modest, and severe level of IPF [9]. New pharmacotherapies based on small molecules and large molecules are also being evaluated for their anti-fibrotic potential. Along the same line, the pathway-directive approach of treating pulmonary fibrosis opened doors for various drugs targeting different proteins such as proteins associated with alveolar epithelial cell dysfunction, ECM remodeling and fibroproliferation and immune dysfunction [10]. For instances, pirfenidone downregulates the expression of α-smooth muscle actin (α-SMA) and fibronectin [11], nindetanib decreases the levels of extracellular matrix proteins [12], collagen 1α1 and fibronectin, tacrolimus suppresses the level of TGF-β1 and related collagen [13], nifuroxazide inhibits Smad/TGF-β pathway [14], and venetoclax/navitoclax downregulates the expression of anti-apoptotic protein, BCL-2/BCL-xL [15], and thereby expression of α-SMA and collagen level [16]. Despites of huge potential of BCL-2 inhibitors in treating fibrosis, their clinical translation and application has been limited by the serious concern related to thrombocytopenia. Therefore, we aim to design and develop a delivery approach for BCL-2 inhibitor, venetoclax (VNT), that can prevent interaction and interfere between the drug molecule and platelets, and facilitate their selective accumulation to the cells of interest [17]. VNT is a BCS Class IV drug and possesses low aqueous solubility with poor permeability [18]. The log P value of VNT is 5.5 and also a substrate for P-gp and BRCP and are primarily metabolized by CYP3A4/5 [19]. These properties make VNT a suitable candidate to be loaded in Liposomes for its efficient delivery in treating IPF.

Prior studies showed that VCAM1 peptide has also been explored as a targeting moiety by attaching it to the nanoparticles for targeting overexpression of VCAM1 protein in inflamed endothelium cells for the treatment of chronic inflammatory disease [20,21]. VCAM1 is a vascular cell adhesion molecule that is a member of the immunoglobulin superfamily [22] and overexpressed in blood vessels after stimulation of cytokines. It also facilitates the adhesion of blood components such as monocytes, lymphocytes, eosinophils, and basophils to vascular endothelium [23]. It has also been reported that VCAM1 is a potential biomarker to predict the mortality in IPF patients [24]. Therefore, we hypothesize that a peptide (amino acid sequence: VHPKQHRGGSKGC) has been shown to possess strong and selective binding affinity with VCAM1 protein can be incorporated with the delivery vehicle for enhancing cell selective accumulation of VNT.

In the present research, VNT-loaded PEGylated LPS Liposomes (VNT-LPS) have been prepared using the Lipids- HSPC, mPEG-DSPE, and cholesterol. Hydrogenated Soy Phosphatidyl Choline (HSPC) is a saturated Lipid used in the preparation of liposomal formulations and has been reported to improve the solubility and bioavailability of poorly soluble drugs [25]. HSPC-based Liposomes possess tighter membrane packing due to their high saturation content and more stability during storage, owing to their less hydrolysis and oxidation [26]. mPEG2000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000]) conjugation to Lipids provides stealth shielding properties and increase circulation time of the formulation in vivo. The hydrophilic portion decreases the interactions of Liposomes with the reticular-endothelial system [27]. Cholesterol is an essential component of the Liposomes drug delivery system as it stabilizes the Liposomes by altering the thickness, fluidity, compressibility, intrinsic curvature, conformation, and redistribution in the bilayer membrane [28]. DSPE-PEG2000-Mal (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol)-2000]) is a self-assembling reagent with reactive maleimide group to bioconjugate targeting molecules such as peptides, antibodies, and protein. Moreover, the functionalization of Liposomes with peptide imparts the selectivity and specificity to the Liposomes, which further assist the drug-loaded Liposomes to reach their target site of action [29].

Various characterization profiles demonstrate that the Liposomes are within 100–120 nm ranges with a partial negative zeta potential and maintain their stability for up to 3 months of preservation at 4 °C. A series of in vitro studies were conducted to evaluate cellular specificity, cytotoxicity, and therapeutic potentials, using lung fibroblast cells. An IPF animal model was prepared upon injecting bleomycin intraperitoneally (IP) and Liposome (VCAM1-VNT-LPS) was injected intravenously to evaluate biosafety and efficacy. The results were compared against non-targeted Liposome (VNT-LPS), free drug (VNT) and untreated IPF control model. The findings from both in vitro in vivo studies demonstrate that VCAM-1 targeted LPS can carry and delivery the payload of VNT to the cells of interest and improve therapeutic outcome in IPF mice with no significant toxicity. Thereby this approach of BCL-2 inhibitor delivery holds a strong and emerging potential of further validation using advanced humanized animal model with long term administration.