Preparation of Doxorubicin-Retinoic Acid Hip Complex Nanomicellar Formulation for Doxorubicin Delivery to Prostate Cancer

Abstract

Doxorubicin is a hydrophilic anticancer drug. Due to hydrophilicity, it’s difficult to encapsulate in a hydrophobic core of nanomicelles. The main purpose of this study was to develop a hydrophobic ion paring complex (HIP) of doxorubicin using hydrophobic retinoic acid (Vitamin A). The resultant hydrophobic (DOX-RA) complex was utilized to prepare drug-loaded nanomicelles by co-precipitation method with penta block copolymer. Nanomicelles (DOX-RA/PBNM), was prepared by evaporation rehydration technique. DOX-RA complex was analyzed by H-NMR and FTIR. The nanomicelles were analyzed for size and zeta potential using dynamic light scattering (DLS) as well as transmission electron microscopy (TEM). The FT-IR and the H-NMR analyses confirmed the structures of the DOX-RA complex. Mean nanomicellar sizes were 25.5nm ± 5.00nm, and ζ-potential was approximately zero. H-NMR and FTIR analysis done on DOX-RA indicate peaks characteristic of both doxorubicin and retinoic Acid. This confirmed successful complex formation. Transmission electron microscopy (TEM) analysis revealed round shaped morphology and sizes similar to DLS results. In vitro release studies revealed that pentablock nanomicelles released Doxorubicin at a slow first order rate in phosphate buffer solution (PBS) at pH 7.4 compared to pH 5.5 and pH 4.0. Confocal microscopy analysis with DOX-RA/ PBNM indicated that pentablock nanomicelles were efficiently taken into prostate cancer (PC3) cells and doxorubicin was efficiently released from the nanomicelles into the cells. In addition, cell proliferation assay showed that nanomicelles ferried adequate amounts of Doxorubicin into PC-3 cells and inhibited cell growth significantly. Results confirm that DOX-RA complex facilitated the encapsulation of doxorubicin within nanomicelles increasing DOX intracellular concentration. Volume 1 Issue 4 Copyrights @ Alex Oselu Owiti. J Urol Neph St Citation: Alex Oselu Owiti. Preparation of Doxorubicin-Retinoic Acid Hip Complex Nanomicellar Formulation for Doxorubicin Delivery to Prostate Cancer. J Urol Neph St 1(4)2019. JUNS.MS.ID.000118. DOI: 10.32474/JUNS.2019.01.000118. 80 In addition, serious cytotoxic effects may be caused to healthy cells due to non-targeted delivery. These may result in dose-dependent cardiac damages, multidrug resistance, and myelosuppression thus, restricting its therapeutic application [5]. It is important to develop new delivery systems that capable of ferrying sufficient amount of drug to the tumor cells, thereby avoiding the multidrug resistance and frequent dose administrations of chemotherapeutics. One of the attempts to elevate the drug bioavailability in tumor tissue is the application of site-specific delivery systems that may release the encapsulated drugs within tumor cells. Another way is to attain higher accumulation of drug by specific tumor cell targeting. However, this may be difficult to achieve without using a ligand that is unique to the target on the cell surface. This lack of proper ligand may end up delivering the drug to the undesired site [6]. Some intrinsic variations in the tumor microenvironment such as enzymes, pH and oxidative stress, and the extrinsic factors i.e., light, temperature and magnetic fields, are known to cause site-specific drug release within the tumor cells and tissues [7]. pH triggered drug release from a nanocarrier is the most acceptable way of drug release directly inside the cell cytoplasm [7]. This strategy of using the acidic tumor microenvironment to trigger drug release has shown some short falls due to the fact that the lowest pH a tumor is located far from tumor blood vessels [8]. Since the interstitial region of a tumor has a pH lower than 6.5, the ability nanocarriers might be restricted. For instance, some carriers such as liposomes become unstable and are altered before arriving at their intended targets. Another promising type of nanoformulation for delivery of hydrophobic drugs is nanomicelles. Nanomicelles display higher efficiency compared to other carriers like liposomes. They also possess many advantages, which include high bioavailability, improved stability of the encapsulated drug, better encapsulation and loading efficiencies and better delayed release profile [9]. Furthermore, most of the materials utilized in its production are biologically safe for all forms of administration. The application of these nanomicelles is with respect to ability to improve drug entry into the tumor tissue, while reducing dosage and enhancing drug efficacy by limiting non-selective cytotoxicity [10-13]. To cause pH-triggered intracellular release of doxorubicin (DOX), retinoic acid (RA), a lipophilic molecule, was utilized to form an ion paired complex (DOX-RA). The formed ion pair shows instability in an acidic microenvironment i.e. pH < 7.0, which is compatible with tumor tissue. The complex attains stability in an environment with pH 7.4, for instance, blood [14]. This would help in increasing the drug levels in the cells of tumor tissue, sparing normal tissues cells. In addition, hydrophobicity caused by ion pairing can retain doxorubicin in the hydrophobic core of nanomicelles and raise the encapsulated amount of DOX in the nanomicelles. Furthermore, studies have revealed that RA may improve effectiveness of a drug by interfering with the permeability and fluidity of membrane of tumor cells [15]. Materials and Methods Retinoic acid was purchased from Aeser, MA, USA. Pentablock copolymer containing Poly-ethylene glycol (PEG), L-lactide and ε-caprolactone monomers, was obtained from our laboratory, already synthesized from previous project. CyquantTM cell proliferation assay kit was obtained from Invitrogen Life Technologies Inc. and distributed by Thermos-Fisher Scientific. Doxorubicin drug was obtained from ADOOQ Bioscience, Irvin, CA, USA. All other reagents utilized in this study were of analytical grade. Preparation of DOX-RA In this study, a hydrophobic complex of doxorubicin and retinoic acid (DOX-RA) was prepared by co-precipitation technique. Briefly, doxorubicin aqueous solution (10 mg/mL), was prepared in an Eppendorf tube, 100mg/mL sodium bicarbonate solution was added to the tube while stirring. An ethanolic solution of retinoic acid (10 mg/mL), was added to the DOX solution while stirring continuously. After stirring the combined solutions for 1 h, the final solution was centrifuged at 6000 rpm for 20 min. Finally, an orange to brick-red colored precipitate of DOX-RA (Figure 1) settled at the bottom of the tube. The precipitated complex was washed 3 times with deionized water, followed by drying at 40 °C before being stored at 4°C. The DOX-RA complex was then used to prepare nanomicelles with pentablock copolymer previously prepared in our lab [16]. Figure 1: The schematic illustration of the reaction and the structures of DOX, RA and DOX-RA complex. Determination of Hydrophobicity DOX-RA Complex Hydrophobicity of DOX-RA complex was analyzed by studying its solubilities in octanol. DOX-RA was dissolved in octanol, while agitating for 48h at room temperature (25°C). The sample was then submitted to centrifugation for 20min at 6000 rpm. After centrifugation, the supernatant was collected and dissolved