Encapsulation and In Vitro Evaluation of Low-Density Lipoprotein with Cholesterol Conjugated Anti-HSP27 and HER2 Proteins as Drug Delivery Enhancement in Ovarian Cancer In Vitro Evaluation of Low-Density Lipoprotein with Cholesterol Conjugated Anti-HSP27 and HER2 Proteins as Drug Delivery Enhancement in Ovarian Cancer.

Context: Low density lipoprotein (LDL) has implications in cancer treatment fields. LDL is a bio-nano particle that is essential for cholesterol transport in the body, which is recognized by cell’s LDL receptors. In ovarian cancer, the LDL receptors are highly expressed more than in healthy normal cells. Therefore, LDL core loading drug targeting dual proteins HSP27 and HER2 through HER2 signaling pathway is an attractive approach for cancer treatment. HSP27 and HER2 are important proteins that are associated with many diseases because of their cellular functions, which are up regulated together in cancer cells more than in benign cells. Objective: The present study was designed to target and evaluate cholesterol conjugated HSP27 and HER2 inhibitor loaded into LDL particles to take the advantages of the metabolic pathways of lipid biochemistry. Materials and Methods: Previous synthesized cholesterol conjugates loaded into commercial LDL core, simply by mixing, sonicating (1:5 ratio) respectively, followed by particles’ size measurements. Then, SKOV3 cells in-vitro growth evaluation using both western blot and MTT assays. Key Finding: Particles’ size was significantly increased after encapsulation of the (cholesterol/Agent 1& 2) into LDL particles. Both cholesterol/Agent 1 and cholesterol/ Agent 2 did not show any precipitation in aqueous solution at both 100 µM and 500 µM. High ovarian cancer cell’s (SKOV3) growth inhibition was shown by western blot assay and MTT assay. Conclusion: LDL encapsulation with cholesterol conjugated anticancer agents proved drug delivery strategy targeting dual proteins which is mimicking the native components of LDL and its metabolic pathway. target HSP27 function in SKOV3 cells and reduce HER2 expression through HER2 pathway.


Introduction
LDL is a vehicle that moves, transports and supply both free and esterified cholesterol through the blood stream to different parts of the body [1]. Lipoproteins are endogenous nanoparticles, structurally consisting of a hydrophobic core that contains cholesteryl esters and triacylglycerol, and the hydrophilic shell contains phospholipids and free cholesterol with one apolipoprotein B-100 for receptor recognition [2]. The particle size of lipoproteins ranges from 10 to 1200 nm, and the size of each type of lipoprotein is as follows: chylomicron, 75-1200 nm; very low density lipoprotein Then, LDL becomes internalized and undergoes the endocytosis process. First, LDL fuses through clathrin coated vesicle, and the receptor will be recycled by the endosome [5,6]. The endosome breaks down the ligand and then the lysosome, the hydraulic enzyme releases and provides the cell with cholesterol cargo which is either used or stored by the cell. The extra cholesterol from the cells is brought back to the liver by high density lipoprotein (HDL) in a process known as reverse cholesterol transport [7,8] as shown in (Figure 1). It has been found that the LDL receptor is over-expressed in numerous cancers more than in normal tissues [9][10][11][12]. Various lipid metabolic pathways, especially those involving fatty acid biosynthesis, along with phospholipids and their enzyme systems, have been found to be involved specifically in ovarian cancer. Ovarian cells under normal physiological conditions rely on the use of endogenously synthesized cholesterol, which is delivered by low density lipoprotein (LDL), but in case of ovarian cancer, cholesterol uptake will be more pronounced because the cells need it for new cell membrane synthesis [13][14][15][16][17].
These findings have led many researchers to investigate the possibility of exploring LDL as a delivery vehicle for cancer diagnostics and therapeutics. Awwad, et al. [18] stated that targeting cancer using cholesterol conjugates to be taken by the natural LDL is considered a perfect drug delivery system for cancer treatment and clinical diagnosis [18][19][20][21]. There are three different ways in which diagnostic or therapeutic agents can be incorporated into LDL. First, drugs can be loaded into LDL via a covalent attachment to the amino acid residues of apolipoprotein Apo-B100 (protein loading). Second, drugs can be loaded through intercalation into the phospholipid's monolayer of LDL (surface loading). Third, drugs can be loaded via substitution of agents into the lipid core of LDL (core loading) [22][23][24]. In general, conjugation of cholesterol to an active agent targets dual proteins HSP27 and HER2 through HER2 pathway, encapsulated into LDL is an attractive approach for cancer therapy (Figure 1). The dual strategies conjugation and encapsulation enhance the ability of the agent to reach and accumulate in the middle of cancer cells without being recognized by the cell's defenses mimicking the endogenous LDL [25][26][27][28]. Therefore, our goal in this present study was an effort to combine two strategies conjugation and encapsulation to target ovarian cancer cells (SKOV3 cell line that is highly expresses HSP27 protein) in preclinical models and to benefit from the lipid metabolism. First, cholesterol conjugated HSP27 and HER2 inhibitor is previously synthesized and characterized [29,30] as an anti-cancer drug. Secondly, loading cholesterol conjugates into commercially available LDL. Agent 1 and agent 2 (
Chemicals and reagents are commercially available and ready for direct use without need for any preparation. Biological investigation experiment was done at Kuwait University.

Western Blot Assay: SKOV3 confluent ovarian cancer cells'
dishes were washed with PBS collected, and harvested by a scraper.

SKOV3 Examination for Drug Potency Using Western Blot Assay Targeting HSP27
In the previous research studies, cholesterol conjugated dual proteins inhibitor was successfully prepared and characterized [29,30]. The potent cholesterol/agents 1 and 2 were chosen for further tests to prove their potency to inhibit the growth of SKOV3 cells. The cell lysates were collected, and we ran a gel electrophoreses experiment to obtain the bands. The data revealed that both agents were potent in targeting HSP27 function and reduce HER2 expression through HER2 pathway based on DMSO control ( Figure 4).

Figure 4:
Western blot assay showed that agents 1 and 2 able to target HSP27 function in SKOV3 cells and reduce HER2 expression through HER2 pathway. and left for 24 hr. to rest, the LDL particle size became 249.6 nm.
Same steps repeated for cholesterol/Agent 2 and the measured particle size of simply mixed sample by hand, was 410.7 nm but sample mixed, sonicated, and left overnight its particle size became 591 nm. Generally, we found that the particle sizes for the mixture of cholesterol conjugates in LDL particles without sonication had small diameters compared to the sonicated samples. This is because after sonication step, LDL particles broken down and to allow the cholesterol/anti-cancer agents to enter it while reforming overnight as shown in (Table 1)   Sonicated samples have larger diameter more than non-sonicated, which proofed the encapsulation.

Declaration of Interest
The author reports no conflict of interest to declare. The author alone is responsible for the contact and writing of this article.

Funding
This study was supported by grant (SC14/18) from the research sector at Kuwait University.