Micellar Nano-Carriers for the Delivery of STAT3 Dimerization Inhibitors to Melanoma
Abstract
The objective of this research was to develop polymeric micellar formulations of inhibitors of signal transducer and activator of transcription 3 (STAT3) dimerization, specifically S3I-1757 and S3I-201, and to evaluate the activity of successful formulations in B16-F10 melanoma, a STAT3 hyperactive cancer model, both in vitro and in vivo. STAT3 inhibitory agents were encapsulated in methoxy poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO114-b-PCL22) and methoxy poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (PEO114-b-PBCL20) micelles using co-solvent evaporation. Polymeric micelles of S3I-1757 showed high encapsulation efficiency, a slow release profile under physiological conditions, and a desirable average diameter for tumor targeting. The same formulations showed low encapsulation efficiencies and rapid drug release for S3I-201. Further studies evidenced the delivery of functional S3I-1757 by polymeric micelles to B16-F10 melanoma cells, leading to a dose-dependent inhibition of cell growth and vascular endothelial growth factor production comparable with that of free drug. Encapsulation of S3I-1757 in polymeric micelles significantly reduced its cytotoxicity in normal bone marrow-derived dendritic cells. Micelles of S3I-1757 were able to significantly improve the function of B16-F10 tumor-exposed immunosuppressed dendritic cells in the production of interleukin-12, an indication for functionality in the induction of cell-mediated immune response. In a B16-F10 melanoma mouse model, S3I-1757 micelles inhibited tumor growth and enhanced the survival of tumor-bearing mice more than free S3I-1757. These findings show that both PCL- and PBCL-based polymeric micelles have potential for the solubilization and delivery of S3I-1757, a potent STAT3 inhibitory agent.
Introduction
Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates the expression of genes involved in cell growth, survival, and differentiation. STAT3 is found to be constitutively active in about seventy percent of human cancers, including malignant melanoma, making a major contribution to cancer growth, survival, metastasis, and immune evasion. Numerous studies have considered STAT3 a valid target for cancer therapy, and several inhibitors of STAT3 have been developed. Nonetheless, clinical advancement of STAT3 inhibitors for cancer therapy has been unsuccessful to date. Poor water solubility and inadequate tumor levels and selectivity have obstructed the clinical development of STAT3 inhibitors in most cases. To overcome these problems, the development of nano-carrier formulations of STAT3 inhibitors has been proposed.
Micellar nano-carriers have been widely used for overcoming the poor water solubility and low tumor selectivity of anticancer drugs. First proposed as drug carriers in 1984, polymeric micelles have since grown to play a significant role in cancer nanotherapy. Polymeric micelles are known to take advantage of the enhanced permeability and retention effect to passively target solid tumors. This can result in increased accumulation of the loaded drug in cancerous tissue and less accumulation in healthy tissues. Other clinical advantages of micelles include their ability to efficiently solubilize hydrophobic drugs within their hydrophobic core and the potential for sustained drug release.
The building block of polymeric micelles is amphiphilic block copolymers. Among the various block copolymers that can be used for the preparation of micelles, poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL)-based copolymers have been the subject of much interest for the solubilization and tumor-targeted delivery of hydrophobic anticancer drugs. The PCL block acts as the hydrophobic core for drug solubilization, and the PEO chains serve as a hydrophilic palisade, protecting the delivery system against opsonization and early elimination by mononuclear phagocytic cells. The safety and biocompatibility of PEO have been established. Biomaterials containing PCL have also been used in a variety of biomedical settings, including osteoarthritis and bone regeneration. Poly-α-benzyl carboxylate ε-caprolactone (PBCL) is a potentially biocompatible derivative of PCL with improved nano-carrier stability when used as a core-forming block of block copolymers. In this study, two recently reported specific inhibitors of STAT3 dimerization, S3I-1757 and S3I-201, were encapsulated in PEO-b-PCL- and PEO-b-PBCL-based polymeric micelles as a strategy for enhancing their water solubility and tumor selectivity. It was hypothesized that polymeric micelles would allow for greater solubilization of STAT3 inhibitors, particularly the derivative with a more hydrophobic structure, S3I-1757, in water, exhibit a slow release of the encapsulated compound over time, and show similar growth inhibitory effects and therapeutic activity to free drug treatments in STAT3-positive B16-F10 melanoma models.
Materials and Methods
Materials
Methoxy PEO was purchased from Sigma-Aldrich, ε-caprolactone was purchased from Lancaster Synthesis, and α-benzyl carboxylate ε-caprolactone was synthesized by Alberta Research Chemicals, Inc. The methoxy PEO had a molecular weight of 5000 Da, corresponding to an average degree of polymerization of 114. S3I-1757 was purchased from Glixx Laboratories and stored according to supplier recommendations; S3I-201 was purchased from AdooQ Bioscience and stored according to supplier recommendations. RPMI media, fetal bovine serum, and penicillin-streptomycin-glutamine were purchased from Gibco via ThermoFisher Scientific.
Cell Line
The murine melanoma cancer cell line B16-F10 of C57BL/6 origin was obtained from American Type Culture Collection. The B16-F10 cells were grown in RPMI-1640 supplemented with ten percent fetal bovine serum, two millimolar L-glutamine, and one hundred IU/mL penicillin/streptomycin at thirty-seven degrees Celsius in a five percent carbon dioxide atmosphere.
STAT3 Binding Studies by Cellular Thermal Shift Assay
A cellular thermal shift assay was performed to determine the strength of binding between the STAT3 inhibitors and their target protein, STAT3. B16-F10 cells were grown in tissue culture-treated plates; when the cell confluency reached over ninety percent, they were treated with three hundred micromolar of free S3I-201, S3I-1757, or equivalent amounts of DMSO for one hour. The cells were then harvested and heated to varying temperatures. The resulting samples were processed and equal amounts of protein were separated on a ten percent SDS-PAGE gel and transferred to a PVDF membrane. The membranes were blocked in five percent non-fat milk and incubated with antibodies against STAT3 and vinculin. The blots were washed, incubated with anti-rabbit or anti-goat IgG, HRP-linked antibodies, and detected with ECL Western Blotting Substrate. The bands were quantified using ImageJ software. For quantification, all bands were normalized to the control protein, vinculin. Normalized values were then taken as a percentage of the values obtained at the lowest temperature, which was forty-nine degrees Celsius in this study.
Polymer Synthesis
Block copolymers (PEO-b-PCL and PEO-b-PBCL) were synthesized by ring-opening polymerization and characterized as reported previously. Briefly, an ampule containing caprolactone or benzyl carboxylate caprolactone, methoxy PEO, and stannous octoate catalyst was vacuum sealed and placed in a one hundred forty degree Celsius oven for four hours. The ampule was stirred occasionally to observe the viscosity of the sample. After the reaction was completed, the ampule was brought to room temperature. Polymer purification was done by washing the product using dichloromethane, hexane, and ether. The mass of the polymer was calculated to determine the yield. Polymer structure was confirmed, and the degree of polymerization of the hydrophobic block was determined by proton nuclear magnetic resonance spectra as mentioned in a previous publication. The degree of polymerization of synthesized PEO-b-PCL was twenty-two and its theoretical molecular weight was seventy-five hundred grams per mole with polydispersity being 1.6. PEO114-b-PBCL20 had a degree of polymerization of twenty and its theoretical molecular weight and polydispersity were ninety-nine hundred sixty grams per mole and 2.1, respectively.
Preparation and Characterization of Micellar Formulations
S3I-1757 and S3I-201 were encapsulated in polymeric micelles using co-solvent evaporation as described before. Briefly, polymers and drugs were dissolved together in acetone until no precipitate remained, then added dropwise to stirring double-distilled water. The solutions were left to stir overnight to allow the acetone to evaporate and micelles to form. The next day, the vial was placed under vacuum for fifteen minutes to remove remaining acetone, if any. The micelle solution was then centrifuged for five minutes at fourteen thousand RPM. All micelle solutions were created in a polymer:drug:water ratio of ten to one to one by mass. Micelle size and polydispersity index were determined with a Zetasizer Nano ZS. Encapsulated drug levels were measured using high-performance liquid chromatography, described below. The encapsulated drug levels were used to calculate the loaded drug content and encapsulation efficiency.
HPLC Method
All samples were run on a Varian Prostar 210 HPLC System using a Varian 335 UV detector at two hundred fifty-four nanometers. A μBondapak C18 analytical column was used. A gradient system with water with 0.1% trifluoroacetic acid and acetonitrile, at a flow rate of one milliliter per minute, was used. For S3I-1757, the mobile phase was programmed as follows: hold at eighty percent water for four minutes, linear gradient from eighty percent to twenty percent water over one minute, hold at twenty percent water for six minutes, linear gradient from twenty percent to eighty percent water over one minute, and hold at eighty percent water for two minutes. The retention time was nine point fifteen minutes. For S3I-201, the mobile phase was programmed as follows: hold at one hundred percent water for one minute, linear gradient from one hundred percent to ten percent water over twelve minutes, and linear gradient from ten percent to one hundred percent water over one minute. The retention time was nine point eleven minutes.
In Vitro Release Study
Free drug at maximum solubility and micellar formulations of S3I-1757 or S3I-201 were prepared and placed into separate dialysis bags. The dialysis bags were sealed and placed in a beaker with three hundred milliliters of phosphate-buffered saline, then in a thirty-seven degree Celsius shaker. At various time points, a one hundred microliter sample of the solutions was taken from inside the dialysis bag, then the volume was restored by adding one hundred microliters of water. To maintain perfect sink conditions, the phosphate-buffered saline was discarded and replaced with fresh solution at each time point. The samples were quantified by high-performance liquid chromatography as described above.
Evaluation of the Inhibitory Effects of Micellar Formulations of S3I-1757 on Cell Growth and Vascular Endothelial Growth Factor Secretion in B16-F10 Cells
The growth inhibitory and cytotoxic effects of different formulations on B16-F10 cells were assessed using the MTT assay. Briefly, a ninety-six well flat bottom plate was seeded such that each well contained ten thousand cells in one hundred microliters of RPMI-1640 media. The cells were left in the incubator overnight to settle at the bottom of the wells. The next day, one hundred microliters of different control and test treatments were added to each well. The plates were then placed in the incubator for twenty-four or forty-eight hours. An MTT solution was added to the treated cells. After three hours, the supernatant was removed, crystals were dissolved in dimethyl sulfoxide, and the plates were read on a plate reader and analyzed via software and Microsoft Excel. The level of vascular endothelial growth factor secreted by B16-F10 cells treated with different formulations was measured using a Quantikine Mouse VEGF ELISA kit according to manufacturer’s instructions.
Assessing the Effects of S3I-1757 Micelles on Bone Marrow-Derived Dendritic Cells
Dendritic cells were obtained from femurs of C57BL/6 mice in complete media in the presence of granulocyte-macrophage colony-stimulating factor as described. Briefly, femurs were removed and purified from surrounding tissue. Both ends of the intact bone were cut, and the bone marrow was flushed with sterile phosphate-buffered saline using an insulin syringe. Leukocytes were triturated and filtered through a forty micrometer cell strainer to obtain a single cell suspension. Bone marrow leukocytes were washed and two million cells were seeded in ten centimeter non-treated cell culture dishes with ten milliliters of dendritic cell complete medium. On day three, another ten milliliters of respective media was added to the plates. On day six, ten milliliters of culture media was replaced with ten milliliters of respective media. By day seven, the cells were ready for use. To determine the toxicity of free and PEO-b-PBCL-encapsulated S3I-1757 to dendritic cells, a twenty-four hour MTT assay was conducted in a similar manner to the B16-F10 MTT assay. Tumor-induced immunosuppressed dendritic cells were generated according to a previously described method with some modifications. To make B16-F10 conditioned media, B16-F10 cells were grown for forty-eight hours, then the supernatants were collected, mixed with dendritic cell complete medium in a one to one ratio, and added to dendritic cell culture on the aforementioned days. Lipopolysaccharide treatment was included as a positive control. The positive control was composed of dendritic cells grown in dendritic cell complete media and given lipopolysaccharide. B16/DC refers to dendritic cells grown in the one to one ratio of B16 conditioned media to dendritic cell complete media. Treatment groups contained either free or PEO-b-PBCL-encapsulated S3I-1757 at five and ten micromolar concentrations. All treatments were exposed to dendritic cells for twenty-four hours. Interleukin-12 was measured in the supernatant collected from the cells.
In Vivo Evaluation of S3I-1757 Micelles in a B16-F10 Melanoma Mouse Model
To evaluate the therapeutic efficacy of S3I-1757 micelles in vivo, a B16-F10 melanoma mouse model was used. Female C57BL/6 mice, aged six to eight weeks, were injected subcutaneously in the right flank with B16-F10 cells suspended in phosphate-buffered saline. Tumor growth was monitored by measuring the length and width of tumors with calipers, and tumor volume was calculated using the formula: volume = (length × width^2)/2. When tumors reached a predetermined size, mice were randomized into treatment groups and administered either free S3I-1757, S3I-1757-loaded micelles, or appropriate control formulations via intravenous injection at scheduled intervals.
Throughout the course of treatment, mice were monitored for changes in body weight, general health, and tumor progression. At the end of the study, or when mice reached humane endpoints, they were sacrificed, and tumors were excised and weighed. The survival of the mice in each treatment group was recorded and analyzed using Kaplan-Meier survival curves to determine the impact of the treatments on overall survival.
Results
Encapsulation Efficiency and Drug Release
Micellar formulations of S3I-1757 demonstrated high encapsulation efficiency, with values exceeding eighty-eight percent, and exhibited a slow release profile under physiological conditions, with less than thirty-two percent of the drug released within twenty-four hours. The average diameter of the S3I-1757-loaded micelles ranged between thirty-three and fifty-four nanometers, a size considered optimal for tumor targeting via the enhanced permeability and retention effect. In contrast, S3I-201-loaded micelles showed lower encapsulation efficiency and a much more rapid drug release, indicating that S3I-1757 is better suited for micellar encapsulation and sustained delivery.
In Vitro Activity
The delivery of S3I-1757 by polymeric micelles to B16-F10 melanoma cells resulted in a dose-dependent inhibition of cell growth, as measured by the MTT assay. The growth inhibitory effect of the micellar formulation was comparable to that of the free drug, indicating that encapsulation did not diminish the drug’s biological activity. Furthermore, treatment with S3I-1757 micelles led to a significant reduction in vascular endothelial growth factor production by B16-F10 cells, suggesting an impact on tumor angiogenesis.
Importantly, encapsulation of S3I-1757 in polymeric micelles significantly reduced its cytotoxicity in normal bone marrow-derived dendritic cells. This reduction in toxicity is a critical advantage, as it may allow for higher therapeutic doses to be administered with fewer adverse effects on normal immune cells.
The ability of S3I-1757 micelles to restore the function of immunosuppressed dendritic cells was demonstrated by the increased production of interleukin-12 in B16-F10 tumor-exposed dendritic cells treated with the micellar formulation. This finding suggests that the micelles not only deliver the drug effectively to tumor cells but also help to counteract tumor-induced immune suppression.
In Vivo Efficacy
In the B16-F10 melanoma mouse model, administration of S3I-1757-loaded micelles resulted in a significant inhibition of tumor growth compared to both free S3I-1757 and control treatments. Mice treated with the micellar formulation exhibited smaller tumor volumes and lower tumor weights at the end of the study. Furthermore, treatment with S3I-1757 micelles led to a marked improvement in the survival of tumor-bearing mice, as demonstrated by Kaplan-Meier survival analysis.
No significant adverse effects or weight loss were observed in mice treated with S3I-1757 micelles, indicating that the formulation was well tolerated. These results support the potential of both PCL- and PBCL-based polymeric micelles as effective nano-carriers for the solubilization and delivery of S3I-1757, a potent STAT3 inhibitory agent.
Discussion
The findings of this study demonstrate that polymeric micellar formulations can successfully encapsulate and deliver S3I-1757, a hydrophobic STAT3 dimerization inhibitor, to melanoma cells both in vitro and in vivo. The high encapsulation efficiency, favorable release profile, and optimal particle size of the micelles contribute to their effectiveness as drug delivery systems. The ability of S3I-1757 micelles to inhibit tumor growth, reduce angiogenic factor production, and enhance the survival of tumor-bearing mice, while minimizing toxicity to normal immune cells, highlights the therapeutic potential of this approach.
Moreover, the restoration of dendritic cell function by S3I-1757 micelles suggests an added benefit in overcoming tumor-induced immune suppression, which is a major hurdle in cancer therapy. By improving the immunostimulatory capacity of dendritic cells, these micellar formulations may enhance the efficacy of immunotherapeutic strategies in melanoma and potentially other STAT3-dependent cancers.
Conclusion
In summary, this research provides compelling evidence that PCL- and PBCL-based polymeric micelles are promising nano-carriers for the delivery of S3I-1757 to melanoma. These formulations offer improved solubility, sustained release, and enhanced tumor targeting, leading to significant anti-tumor activity and improved survival in preclinical models. The reduced toxicity to normal dendritic cells and the restoration of immune function further support the potential clinical application of S3I-1757 micelles in the treatment of STAT3-driven malignancies.