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

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


Introduction
Doxorubicin (DOX), available in the market as Adriamycin or Rubex, is a drug used in treatment of various cancers. Doxorubicin is a member of anthocyanins family and is grouped under anthracycline with both antitumor and antibiotic activities. It works by interfering with DNA function causing intercalation and alkylation of DNA, causing disruption to both RNA and DNA polymerase, inhibition of topoisomerase II which consequently kills the tumor cells [1,2]. DOX is applied mostly in the treatment of breast and bladder cancers. It is also used in cases of sarcomas, lymphomas, and acute lymphatic leukemias. It is normally used as combination with other drugs by intra-venous application.
However, there are many serious side effects associated with doxorubicin [3]. These side effects include but not limited to allergic reactions which may cause tissue damage at the spot of injection. Another most notorious side effect is the accumulation of doxorubicin in the cardiac and respiratory tissues. This irreversible dose-dependent, side effect may cause toxicity to cardiac and respiratory tissues, resulting in cardiomyopathy, dyspnea and intolerance to exercise due to the production of mitochondrial reactive oxygen species (ROS). Other common collateral effects are loss of hair, suppression of bone marrow, skin eruptions, vomiting and mouth inflammation. Patients often experience red coloration of the urine for a few days during treatment with DOX. Doxorubicin is a widely used drug in clinical setting. However, less entry and low distribution of doxorubicin in tumor tissue are the principal factors for its therapeutic backlash [3]. Due to low pH in the tumor interstitial environment, weak base drugs like doxorubicin, are likely to ionized before entering leading to reduced cell uptake [4].
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][11][12][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. 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].

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 in isopropanol, homogenized and then analyzed using UV-vis spectrophotometer (Shimadzu, Kyoto, Japan) 495nm wavelength.

FTIR Analysis of DOX-RA Complex
To determine structural characteristic of the formed, DOX-RA complex, and the success of the complex formation. Samples of synthesized DOX-RA complex, RA and DOX were subjected to FT-IR, Thermo-Scientific Nicolet iZ10, with an ATR diamond and DTGS detector. Where samples of DOX-RA complex, DOX and RA were analyzed at scanning range of 650-4000 cm −1 .

Mass Spectrometry Analysis for DOX-RA complex
The DOX-RA complex was submitted to mass spectrometry (Thermo LCQ deca mass spectrometer). The analysis with LC-MS/ MS involved a positive ion mode with electrospray ionization (ESI) as the main ion source. The source was set at 5 kV, the rates of both sheath gas and auxiliary gas flow were set at 80 and 20 units, respectively while capillary temperature was adjusted to 250 °C.
C-18 column of 2.0mm × 50mm, and particle size limit 3 μm was used for Liquid Chromatography separation applying using water with 0.1% formic acid as the mobile phase A and acetonitrile as the mobile phase B. The flow rate of LC was adjusted to 0.20 ml/min.
The gradient of mobile phase B increased from 30% to 100%.

Preparation of DOX-RA Nanomicelles
Nanomicelles(NM) were prepared using hydration-dehydration method [17]. Briefly, a specific amount (0.1%w/v) of DOX-RA complex was dissolved in 1ml of ethanol in an Eppendorf tube. On the other hand, 2%w/v of pentablock copolymer was dissolve in 1 mL ethanol in a separate tube. The two solutions were then mixed together, homogenized and evaporated in a speed vacuum for 24h until the formation of a thin film. Resultant thin film was suspended in deionized (DI) water followed by vortexing until a complete dissolution was achieved [18]. The solution was filtered using 0.22µm filter membrane to acquire uniform nanomicelle sizes and eliminate the polymer residues. The nanomicelle suspension was further freeze-dried using LABCONCO freeze-dryer, USA, with trehalose as cryo-protectants [2].

Determination of Nanomicelle Size and Zeta Potential
The sizes, zeta potentials and polydispersity indices (PDI) of DOX-RA-PBNM were obtained using dynamic laser scattering (DLS) with the help of Zetasizer HS 3000 (Malvern Instruments, UK), at a detection angle of 90 degrees at 25 o C. The samples were dissolved in deionized water, homogenized and filtered using 0.22µL before being transferred into a transparent cuvette for analysis. Average values of three measurements were obtained for all samples and the peaks for zeta sizes, PDI and zeta potentials were obtained and recorded accordingly [19].

Determination of Drug Encapsulation and Loading Efficiencies
The amount of doxorubicin encapsulated in the nanomicelles was measured by UV-vis. briefly, a portion of the nanomicellar formulation was freeze-dried. Then 2mg of the dry formulation was  were reported in parts per million (ppm) [21]. 1H-NMR spectra were recorded from 0 to 12 ppm using a delay time of 4 seconds. Briefly; a small amount of freeze-dried DOX-RA-loaded pentablock nanomicellar formulations were dissolved in DI water to form aqueous solution. The samples were then stained with 1% uranium salt. The resultant solution containing nanomicelles was then placed on a carbon-coated copper grid, and excess liquid was removed using a piece of dry filter paper then submitted to transmission electron microscope.

In Vitro Release Study for DOX-RA Nanomicelles
The release of doxorubicin from DOX-RA/PBNM was conducted in various media of different pH conditions (pH 4.0, pH5.5 and pH 7.4) for 108 h in a dark room [22]. Briefly, 2mL of (2%w/v), DOX-RA/PBNM aqueous solution was transferred into a dialysis membrane bag (MW 10kDa) [23]. The dialysis bags were dipped

Hydrophobicity Analysis for DOX-RA Complex
Hydrophobicity of DOX-RA complex was analyzed before the preparation of DOX-RA nanomicelles. The lipid solubility of saturated DOX-RA was measured in octanol in order to assess the impact of ion pairing on lipophilicity of doxorubicin [28].

Mass Spectrometry Analysis for DOX-RA Complex
Mass spectrometry was conducted to confirm the constitution of the DOX-RA complex with the above-mentioned MS/MS conditions.

Size Distribution and Zeta Potential for DOX-RA Loaded Nanomicelles
Solvent evaporation method was applied for the preparation of DOX-RA/PBNM. The PBNM formulations were analyzed to determine the size, polydispersity index (PDI), and zeta-potential.
Average mean diameters for the nanomicelles (DOX-RA/PBNM) was 25.5±5.00nm (Figures 3 & 4). Small negligible peaks appeared at regions beyond 1000nm for DOX-RA/PBNM, perhaps due to a small aggregation in the sample, but still the majority peak (around 98%) was 25.5±5.00nm ( Figure 5A). Average PDI for DOX-RA/ PBNM 0.037±0.005. PDI values suggest that the nanomicelles exhibit uniform particle size distribution with less aggregation. The drug-loaded nanomicelles display neutral zeta potential (around 0±2.5 mV) as shown in Figure 5B. This makes it possible for the DOX-RA nanomicelles to be an excellent candidate for intravenous application since it is unlikely to produce interactions with blood components due to neutrality.  The morphology of pentablock nanomicelles containing DOX-RA complex was investigated by transmission electron microscopy (TEM). This analysis showed that the nanomicelles were spherical, homogenous, and did not contain aggregates ( Figure 6). Regarding size, pentablock nanomicelles had an average of 30±2.5nm. There was an apparent uniform morphology in DOX-RA/PBNM, with uniform appearance. The particle sizes visualized in the TEM images corroborate with the sizes obtained by DLS which already depicts the DOX-RA/PBNM and the empty PBNM sizes as 30nm ±5.00nm.

In Vitro Release Study for DOX-RA-PBNM
The drug release profile of doxorubicin from DOX-RA/  [29].
However, in this study, the release profile in low pH media (pH 5.

In Vitro Cell uptake studies of DOX-RA/PBNM
Cell uptake study was conducted to ascertain the presence of DOX inside the cells after exposure to the Nanomicelles. DOX-RA loaded pentablock nanomicellar formulation was incubated with Prostate cancer PC-3 cells for 12 h and 24 h under conducive conditions of 5% carbon dioxide, 37 o C (30). An appropriate media for the growth of the cells was used [30][31][32]. Cells were incubated in chamber slides with pentablock nanomicelles bearing DOX-RA complex [33,34]. The results of this study showed that there was a significant uptake of the nanomicelles into prostate cancer (PC-3) cells, with higher amount of drug entering the cells after 24 h compared to 12 h as shown in Figure 8 [35][36][37]. In Figure 8B, brickred DOX was observed with high intensity all over the cytoplasmic region including the nucleus. Therefore, it can be concluded that fluorescent DOX molecules are inside the cells. This indicates that nanomicelles were internalized by PC-3 cells. This is an evidence that a large amount DOX was taken inside the cells with DOX-RA-PBNM and the release was maximum afterv24 h ( Figure 8B). The intensity of fluorescence is therefore directly proportional to the amount of DOX that enter the cells. Figure 8 shows a confocal image of PC-3 cells observed after incubation with DOX-RA/PBNM for 12 h and 24 h. The fluorescence intensity within cells treated with cytoplasm. There was a significant amount of DOX-RA-loaded nanomicelles inside the cytoplasm (p < 0.05). This observation suggests that high uptake of the drug loaded DOX-RA-PBNM nanomicelles may have been due to endocytosis. The uptake and distribution of DOX-RA/PBNM by PC-3 cells that was observed by confocal microscopy could be related to the anti-tumor efficacy of DOX. Since DOX itself is fluorescent, it could be applied to directly measure cell uptake without applying any fluorescent marker.  After 48 h, it was observed the cell survival decreased with the increase in drug concentration. The cytotoxicity of DOX was almost the same as that of DOX-RA/PBNM at 24 h time point. However, depending on the increase in concentration of DOX-RA/PBNM, cytotoxicity continuously increased to a level much greater than that of pure doxorubicin, perhaps as a result of the delayed release of doxorubicin from pentablock nanomicelles (Figure 9). In general terms, long-term treatment with a drug where concentration gradually increases is much safer than an abrupt supply of high drug level [39]. This study demonstrates that delivery systems such as DOX-RA/PBNM may lower cytotoxicity or side effects caused by chemotherapy involving doxorubicin [6,39].