Ciprofloxacin derivative‐loaded nanoparticles synergize with paclitaxel against type II human endometrial cancer

Abstract

Combinations of chemotherapeutic agents comprise a clinically feasible approach to combat cancers that possess resistance to treatment. Type II endometrial cancer is typically associated with poor outcomes and the emergence of chemoresistance. To overcome this challenge, we developed a combination therapy comprised of a novel ciprofloxacin derivative-loaded PEGylated polymeric nanoparticles (CIP2b-NPs) and paclitaxel (PTX) against human type-II endometrial cancer (Hec50co with loss of function p53). Cytotoxicity studies revealed strong synergy between CIP2b and PTX against Hec50co, and this was associated with a significant reduction in the IC_μof PTX and increased G2/M arrest. Upon formulation of CIP2b into PEGylated polymeric nanoparticles, tumor accumulation of CIP2b was significantly improved compared to its soluble counterpart; thus, enhancing the overall antitumor activity of CIP2b when co-administered with PTX. In addition, the co-delivery of CIP2b-NPs with paclitaxel resulted in a significant reduction in tumor progression. Results also indicated that NPs significantly increased the CIP2b concentration in tumors compared to the soluble form. Histological examination of vital organs and blood chemistry was normal, confirming the absence of any apparent off-target toxicity. Thus, in a mouse model of human endometrial cancer, the combination of CIP2b-NPs and PTX exhibits superior therapeutic activity in targeting human type-II endometrial cancer.

Graphical Abstract

CIP2b synergizes with paclitaxel to combat resistant endometrial cancer. CIP2b-loaded nanoparticles (CIP2b NPs) showed several-fold higher intracellular uptake in endometrial cancer cells in vitro, and higher intra-tumoral levels of CIP2b in vivo, compared to unencapsulated CIP2b. CIP2b NPs and paclitaxel significantly slowed endometrial cancer tumor growth in mice, compared to paclitaxel with drug-free nanoparticles, or paclitaxel with free unencapsulated CIP2b.

1. INTRODUCTION

In contrast to most other types of cancer, the incidence and mortality rates among women with endometrial cancer (EC) are rising in the United States [1–3]. EC is typically categorized into either type I or type II, with the latter being less common but more aggressive. Patients with type II EC exhibit an increased risk of metastasis and relapse, poor prognosis, and higher mortality rates [1]. More than 80% of type II EC cases exhibit inactivating p53 mutations (loss of function (LOF) p53). EC cells expressing LOF p53 can maintain the G2/M checkpoint in the cell cycle by activating the compensatory anti-apoptotic P38/MK2 pathway and its downstream components, which ultimately desensitizes cancer cells against chemotherapies. To overcome the emergence of chemo-resistance, and to combat the heterogeneity of tumors, chemotherapeutic cocktails are used. For type II EC, a combination of paclitaxel (PTX) and carboplatin is common. However, PTX itself is associated with several side effects including peripheral neuropathy and myelosuppression, both of which are dose-related and dose-limiting [4–7].

Severe peripheral neuropathy occurs in up to 30% of patients receiving taxanes (especially PTX) [6] and is sometimes associated with long-term irreversible functional disability [7, 8]. Such irreversible adverse events result in a decrease in the quality of life for the patient and impose an economic burden on the health system (e.g., bone fractures resulting from increased incidence of falls). On the other hand, even with the co-administration of expensive prophylactic G-CSF treatment courses, PTX doses may need to be reduced as a result of neutropenia or thrombocytopenia. [9]. Using a relatively non-toxic adjuvant has the potential to enhance the quality of life of cancer survivors by reducing PTX-related, dose-dependent adverse events.

We previously reported synthetically lethal NP formulations of PTX and the triple angiokinase molecular inhibitor BIBF 1120 (also known as nintedanib) whose synergistic combination abrogated the restored G2/M checkpoint in LOF p53 EC cells in vitro and in vivo [10]. BIBF1120 inhibits vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), and restores the chemo-sensitivity of the human LOF p53 endometrial cancer cells towards PTX via bypassing the G2/M checkpoint, thus forcing the cells to go through mitotic catastrophe, achieving synthetic lethality. Synergy between PTX and BIBF1120 together with improved tumor accumulation due to the enhanced permeation and retention (EPR) phenomenon contributed to create the synthetically lethal ‘nano-bullet’ that markedly inhibited the growth of human endometrial cancer xenografts (LOF p53) in nude mice [11]. Furthermore, we later developed NPs loaded with the MEK1/2 inhibitor PD98059, which synergized with PTX against BRAF^V600E melanoma [12]. In this report, we describe a recently formulated derivative of the fluoroquinolone antibiotic ciprofloxacin (CIP), CIP2b, which previously showed moderate cytotoxic activity against various cancer cell lines [13]. When tested against human EC cell lines, this compound is also capable of synergizing with PTX to augment its efficacy by inducing cell death [14]. Our study showed that CIP2b is safe and effective and synergizes with PTX to halt the progression of LOF p53 type II EC in vitro and in vivo. Encapsulating CIP2b in PEGylated polymeric nanoparticles (NP) further improved the overall antitumor activity of PTX and minimized off-target effects. These findings hold the promise of a combined therapeutic strategy to treat type II EC.

2. METHODS

2.1. Compounds and reagents

Amicon^® Ultra-15 Centrifugal Filter Unit, Ultracel-100 regenerated cellulose membrane (molecular weight cutoff 100 kDa) (Cat. No. UFC9100), TPGS (Cat. No. 57668), dimethyl sulfoxide (DMSO, Cat. No. D2438), and Float-A-Lyzer G2 Dialysis Devices, 8–10 kD (Cat. No. G235025) were purchased from Sigma Aldrich (St. Louis, MO). Ciprofloxacin (Cat. No. 449620050), NP-40 Surfact-Amps^™ 10% Detergent Solution (Cat. No. 28324), Oregon Green^™ 488 PTX (PTX-OG, Cat. No. P22310), propidium iodide (PI) 1 mg/mL (Cat. No. P3566), RNase A (DNase and protease-free) 10 mg/mL (Cat. No. EN0531), and Tween-80 (Cat. No. BP338–500) were purchased from Thermo-Fisher Scientific (Waltham, MA). The CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS) (Cat. No. G3581) was purchased from Promega (Madison, WI). Paclitaxel powder (Cat. No. P-9600) was purchased from LC Laboratories (Woburn, MA) while paclitaxel (6 mg/mL, Cat. No. 70860-200-05) for intravenous (IV) administration was purchased from Athenex, Inc. (Buffalo, NY). Poly(D,L-lactide-co-glycolide) 50:50 (PLGA, RESOMER^® RG 502H) was purchased from Evonik Industries AG (Darmstadt, Germany). All other reagents, buffers, and solvents were at least of analytical grade and were used as received without further purification.

2.2. Synthesis and physicochemical characterization of CIP2b

CIP2b was synthesized and purified as previously described [13, 14]. The UV-Vis absorbance spectra of CIP2b and ciprofloxacin (CIP) were measured at 50 μg/mL in methanol using a SpectraMax Plus 384 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) in scanning mode. For Fourier-transform infrared spectroscopy (FT-IR) analysis, KBr was dried in an oven overnight to remove moisture. CIP2b or CIP was mixed separately with dried KBr and ground into a fine powder using a mortar and a pestle. The translucent KBr pellet was formed by compressing the powder into a small disc. The FT-IR signal between 4000 to 450 cm⁻¹ was captured using PerkinElmer Frontier^™ Infrared Spectroscopy (FTIR) with 64 scans and 1 cm⁻¹ resolution (Perkin Elmer, San Jose, CA). In addition, the solubility of CIP2b in different organic solvents and solubilizers was determined using a small-scale shake flask method using 14 different organic solvents and solubilizers [15]. An excess amount of the drug was added to 0.5 mL solvent in an amber tube and shaken for 48 h at room temperature (RT). Solubility was determined by HPLC following 24 h equilibration at RT and centrifugation at 16000 xg for 10 min and then the supernatant was harvested for measurement. An HPLC system (Agilent Infinity 1100, Santa Clara, CA) equipped with a Waters Symmetry RP-C18 column (5 μm pore size, 4.6 mm × 150 mm, Milford, MA) was used in this experiment. Furthermore, the solubility of CIP2b in aqueous solutions of varying pH was determined by the shake flask method where an excess amount of the drug was added to 5 mL of aqueous buffer with pH values ranging from 2 to 12 and shaken for 72 h followed by 24 h equilibration and centrifugation at 16000 xg for 10 min and then the supernatant was harvested for measurement. All buffers were prepared at 20 mM concentration and constant ionic strength of 100 mM was maintained by adding NaCl as required. The buffer system was chosen based on their pKa values, as follows: for pH 2, a maleate buffer system was used; for pH 3, 4, 5, and 6, a citrate buffer system was used; for pH 7 8, and 12, a phosphate buffer system was used; for pH 9, a tris buffer system was used; and for pH 10 and 11, a methylamine buffer system was used. Finally, the solubility was determined by HPLC. In addition, solubility in water without any buffer system was also determined in a similar manner.

The octanol-water partition coefficient (K_oct/water) of CIP2b was measured following a previously published method with minor modifications [15], and the pKa was determined using the solubility formula (Equation 1) where the intercept = (log S_o – pKa) and slope = 1. Briefly, octanol was saturated with DPBS for 24 h followed by equilibration for 1 h at RT. CIP2b was dissolved in DPBS-saturated octanol at 3 mg/mL and CIP2b concentration in octanol was measured by HPLC (C₁). Octanol containing CIP2b was mixed with DPBS at a 1:20 ratio (Octanol:DPBS) and kept in a horizontal orbital shaker for 16 h at 250 rpm at RT. Following 1 h equilibration at RT, partitioned samples were centrifuged at 16000 xg for 10 min, and the supernatant was analyzed by HPLC to determine CIP2b concentration (C₂). K_oct/water was determined using Equation 2 where C₁: CIP2b concentration in octanol, C₂: CIP2b concentration in octanol after partitioning, V_PBS: Volume of PBS in the partition, V_oct: Volume of octanol in the partition. Intrinsic solubility, which refers to the equilibrium solubility of an ionizable compound at a pH where it is fully unionized, was also determined. pKa was determined by the solubility method. Solubility changes as a function of pH give the basis for determining the pKa of the drug. A sharp decline in solubility is expected at pH values near or greater than the pKa value [16, 17].

$${\text{log}\text{S}=\text{log}\text{S}}_{\text{o}}+\text{log}\left({10}^{–\text{pka}+\text{pH}}+1\right)=\left({\text{log}\text{S}}_{\text{o}}–\text{pKa}\right)+\text{pH}$$ Equation 1

$${\text{K}}_{\text{oct}/\text{water}}=\left({\text{C}}_1/\left({\text{C}}_1-{\text{C}}_2\right)\right)*\left({\text{V}}_{\text{PBS}}/{\text{V}}_{\text{oct}}\right)$$ Equation 2

Moreover, chemometric analysis of CIP2b was conducted on Chemicalize by ChemAxon to predict physicochemical properties. Chemicalize evaluated drug-likeness by testing CIP2b against Lipinski’s rule of five to check if there is any violation of the rules. Chemicalize was also used to predict acidic and basic pKa, isoelectric point, log K_oct/water (also called log P), and intrinsic solubility. SwissADME was used to predict topological polar surface area (TPSA), log P, and oral absorption predictability [18].

2.3. Cytotoxicity assay

Hec50co cells, a subline of Hec50 cells, with lost p53 expression are a poorly differentiated human EC cell line and are widely used in the investigation of EC. Hec50co were cultured in Dulbecco’s modified Eagle’s medium 1X (DMEM, Cat. No. 11965–092) supplemented with 1% penicillin/streptomycin (Pen/Strep, 100 U/mL, Cat. No. 15140122) and 10% fetal bovine serum (FBS, Cat. No. S11150) (ThermoFisher Scientific). Cytotoxicity experiments were carried out using the MTS assay [14, 19–22]. In brief, Hec50co cells were seeded at 1500 cells per well in 96-well tissue culture plates for 24 hours in a complete medium. The medium was removed and PTX, CIP2b, or CIP (all dissolved in DMSO and diluted in complete medium) were added to cells as either monotherapies or in the indicated combinations, and the plates were incubated for 72 hours. Subsequently, the complete medium was removed and the MTS reagent in the complete medium was added to the wells. Plates were incubated for 1–4 hours according to the manufacturer’s protocol. Finally, the absorbance was recorded at 490 nm using a Spectra Max plus 384 Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA). Percentage cell viability was expressed as the average absorbance of the test group relative to that of the control group (untreated cells). The concentration that inhibited cell proliferation by 50% relative to the control (IC₅₀) was calculated using GraphPad Prism (La Jolla, CA). Synergism was detected following the Chou-Talalay method (CI < 1 indicates synergism) [23] using CompuSyn software (Ver. 1.0).

2.4. Cell cycle assay

Hec50co cells were seeded at 250 × 10³ cells per well in 6-well plates for 48 hours in the complete medium. The medium was then removed and replaced with PTX (1, 5, and 10 nM), CIP2b (10 μM), or combinations of PTX with CIP2b, or with a fresh complete medium. Cells were incubated with the treatments for 24 hours, then the medium was removed, and the cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS 1X, ThermoFisher Scientific), trypsinized (0.4 mL/well), and collected by centrifugation (230 xg, 5 minutes). Cells were fixed using ice-cold 70% ethanol and stored at 4°C for 30 minutes, then incubated with 100 μg/mL RNase A in 2% v/v NP-40 Surfact-Amps^® solution (0.5 mL/sample) for 30 minutes at 37°C. PI (50 μg/mL, 0.5 mL/sample) was added prior to analysis by flow cytometry (FACScan, BD Biosciences) using CellQuest software (Ver. 3.3). The data were further analyzed by ModFit LT (Ver. 5.0) to calculate the percent of cells in each phase of the cell cycle (i.e., G0/G1, S, G2/M) [24].

2.5. Preparation of the CIP2b-loaded PLGA nanoparticles

CIP2b-loaded PLGA nanoparticles (CIP2b-loaded NPs) were prepared using a nanoprecipitation technique that involves the precipitation of PLGA/drug from a partially water-miscible organic solution and the diffusion of the organic solvent in the aqueous medium in the presence of TPGS. Briefly, CIP2b and PLGA were dissolved in a mixture of acetone and methanol. This solution was added dropwise into a stirring TPGS aqueous solution in UltraPure^™ water (DNase/RNase-free distilled water). After CIP2b-loaded NPs were formed, the colloidal suspension was transferred to a round bottom flask, and the organic solvents were removed under vacuum using a rotary evaporator (RotaVap R300, Buchi Labortechnik AG, Switzerland). The surfactant was removed using Amicon^® ultra-15 centrifugal filter units following centrifugation and washing with UltraPure^™ water. CIP2b-loaded NPs were finally collected and used directly. Blank (drug-free) NPs were prepared using the same method but without adding the drug.

2.6. Characterization of CIP2b-loaded PLGA nanoparticles

CIP2b-loaded NPs or blank NPs were diluted 100-fold with NanoPure^™ water, and the particle size and zeta potential were determined using a Malvern Zetasizer Nano ZS (Malvern Panalytical, Westborough, MA). Also, a Hitachi S-4800 scanning electron microscope (SEM, Hitachi High-Technologies, Ontario, Canada) was used to examine the shape and surface morphology of the prepared CIP2b-loaded NPs. First, a droplet of the diluted NP suspension was placed onto a silicon wafer on an aluminum stub and was left to air-dry for 24 hours. It was then sputter-coated with iridium using an EMS150T-ES sputter coater (Quorum Technologies, Lewes, UK) and scanned using the SEM operated at 5kV accelerating voltage [12].

2.7. CIP2b content measurement

Briefly, 10 μL of the NP suspension was dissolved in 90 μL acetonitrile, then 0.9 mL methanol was added, and the solution was sonicated for 30 seconds. One mL of NanoPure^™ water was added and the solution was centrifuged (16,000 xg, 5 minutes) and injected directly into the HPLC for content measurement. CIP2b content (μg CIP2b/mL NP suspension) was calculated using Equation 3. An HPLC system (Agilent Infinity 1100, Santa Clara, CA) equipped with a Waters Symmetry RP-C18 column (5 μm pore size, 4.6 mm × 150 mm, Milford, MA) was used in this experiment.

$$\text{Drug}\text{content}=\text{content}\text{of}\text{CIP}2\text{b}\text{in}\text{the}\text{final}\text{formulation}\left(\mu \text{g}\right)/\text{the}\text{total}\text{volume}\text{of}\text{the}\text{CIP}2\text{b}\text{NP}\text{suspension}\left(\text{mL}\right)$$ Equation 3

2.8. In vitro release

CIP2b-loaded NPs (containing 120 μg of CIP2b) were diluted in NanoPure^™ water to a total volume of 1 mL and added to pre-rinsed Float-A-Lyzer G2 tubes. The Float-A-Lyzer tubes were placed in 12 mL of the release medium (DPBS 1X, pH 7.4 with 0.4% w/v Tween-80) inside a 50-mL Falcon tube (n = 3). The tubes were incubated at 37°C and shaken at 300 rpm in an orbital incubator-shaker (New Brunswick Scientific, Edison, NJ). At pre-determined time points, the whole volume inside the Falcon tubes was removed and replaced with a fresh medium. CIP2b content in the samples was measured as described above.

2.9. In vitro intracellular accumulation of CIP2b

Hec50co cells were seeded at a density of 200,000 cells per well in 6-well plates in a complete medium and incubated for 24 hours. Afterward, the medium was removed, and treatments (CIP2b in DMSO or CIP2b-loaded NPs) were added at 100 μM CIP2b per well, and the final volume was made up to 2 mL with the medium. Cells were incubated with the treatments for 2 hours, after which, the medium containing treatment was removed, and the cells were thoroughly washed twice with 1x DPBS. Cells were trypsinized and collected in 15 mL tubes and centrifuged at 230 xg for 5 minutes. The supernatant was aspirated while cell pellets were washed twice with 1x DPBS. Finally, cell pellets were resuspended in 1 mL methanol, and samples were placed in a shaker overnight. Subsequently, samples were centrifuged, and the supernatants (methanol containing free CIP2b) were collected and analyzed using HPLC.

2.10. In vitro activity of CIP2b-loaded NPs

The intracellular accumulation of PTX co-administered with CIP2b-loaded NPs was investigated. CIP2b-loaded NPs were added at 4 or 40 μM, either alone or in combination with PTX-OG (400 nM). An equivalent quantity of blank NPs was also added, either alone or with PTX-OG. Samples were analyzed using a flow cytometer, as described above. In addition, the cytotoxic activity of the CIP2b-loaded NPs and blank NPs was tested against Hec50co cells following the same procedure described above. The CIP2b equivalent concentration of CIP2b-loaded NPs was added instead of the CIP2b solution. An equivalent quantity of blank NPs was also tested to account for the effect of the PLGA NPs alone without CIP2b. Similarly, cell cycle analysis was tested following the incubation of Hec50co cells with CIP2b-loaded NPs instead of CIP2b solution, as described above.

2.11. Animals for in vivo Experiments

For experiments involving the human endometrial cancer xenograft model (Hec50co LOFp53), female athymic Nu/Nu mice (6–8 weeks, Charles River, Wilmington, MA) were used. For other experiments (e.g., pharmacokinetics, biodistribution, safety), female and/or male BALB/c mice (6–8 weeks, Jackson Laboratory, Bar Harbor, ME) were used. Mice were kept under controlled temperature (23 ± 2°C) at the University of Iowa animal care facility. Food was provided ad libitum, and mice were exposed to 12 hours of light/dark cycles. All animal experiments performed were approved by the University of Iowa Institutional Animal Care and Use Committee (IACUC), animal protocol No. 8012099. When anesthesia was needed, mice were injected IP with the ketamine-xylazine mixture (87.5 mg/kg ketamine and 12.5 mg/kg xylazine) prior to performing the experiment.

2.12. Pharmacokinetics and biodistribution

Female BALB/c mice (6–8 weeks, Jackson Labs, Sacramento, CA) were injected IV via the tail vein with either 1.25 mg/kg or 3.75 mg/kg CIP2b-loaded NPs in DPBS. Due to the lack of information about the CIP2b pharmacokinetics and exposure, two doses were studied; a low dose (1.25 mg/kg) and a relatively high dose (at least 2-fold higher) of 3.75 mg/kg. After pre-determined time points (5, 15, 30, 60, 180, 300, and 1440 minutes), mice were euthanized using CO₂ followed by cervical dislocation, and their blood was collected by cardiac puncture in heparinized tubes. Blood samples were centrifuged at 16,000 xg for 15 minutes at 4°C, and plasma was collected. Two mL ethyl acetate and 15 μL of the internal standard (IS) solution (50 μg/mL of 7-OH flavone in methanol) were added to 200 μL plasma, and the mixture was vortexed for 5 minutes, centrifuged (16,000 xg, 5 minutes), and separated. The process was repeated, and a total of 4 mL ethyl acetate per sample was completely evaporated under a steady nitrogen stream using TurboVap LV (Caliper LifeSciences) at room temperature for 90 minutes. Next, 150 μL of methanol and water mixture (1:1) were added to the residue, and the resultant dispersion was centrifuged (16,000 xg, 30 minutes). Finally, the clear supernatant was injected into the HPLC for CIP2b quantification. Also, organs (liver, spleen, lungs, heart, kidneys, and pancreas) were collected from each mouse employed in the pharmacokinetics experiment and were stored at −80° C. To extract the drug, thawed organs (on ice) were homogenized for 120 seconds at speed #5 using a bead tissue homogenizer (Fisherbrand^™ Bead Mill 4 Homogenizer) in screw-capped 2 mL tubes each containing ~25 zirconia/silica disruption beads and 0.25 mL of DPBS. Subsequently, 2 mL ethyl acetate and 15 μL IS solution were added to the tissue homogenate, and CIP2b was extracted twice similar to the procedure described above with the plasma. Next, the total volume (4 mL) of ethyl acetate was evaporated to dryness under a vacuum, and the residue was reconstituted as described in the PK study. Finally, the clear supernatant was injected into the HPLC for CIP2b quantification. Pharmacokinetics data were analyzed using PK Solver Excel Add-in (non-compartmental analysis) to decipher the basic parameters. Furthermore, organ data were used to reproduce fitted models (1-compartment) of organ CIP2b concentrations vs. time plots following CIP2b or CIP2b NPs injection.

2.13. In vivo tumor efficacy and safety study of the drug combination

Female athymic Nu/Nu mice (6–8 weeks old, Charles River, Wilmington, MA) were subcutaneously challenged with Hec50co cells (1 × 10⁶ cells/mouse). When mice had palpable tumors (day 18 post-tumor challenge), they were randomly distributed into 4 treatment groups: PBS (n=6), PTX + blank NPs (n=7), PTX + CIP2b solution (n=7), and PTX + CIP2b-loaded NPs (n=7). Treatments were administered by IV injection in the tail vein in three doses on days 19, 23, and 26 post-tumor challenge. Each dose consisted of 200 μL of either PBS, 5 mg/kg PTX (Paclitaxel concentrate diluted in DPBS) + blank NPs (equivalent to the amount of PLGA present in mice that received CIP2b-loaded NPs), 5 mg/kg PTX + 5 mg/kg CIP2b (CIP2b in DPBS:Tween 80:ethanol 85:10:5; further dilution was made using DPBS), and 5 mg/kg PTX + CIP2b-loaded NPs (equivalent to 5 mg/kg CIP2b). Treatments that included CIP2b-loaded NPs or blank NPs were diluted with DPBS to the required volume. Body weights and tumor volumes (using Equation 4) were monitored twice a week for up to 4 weeks after the last dose (i.e., up to day 50 post-tumor challenge). On the last day of the study (day 50 post-tumor challenge), mice were euthanized, tumors were collected, and their weights were evaluated. Tumor specimens were processed into paraffin tissue blocks followed by sectioning and mounting onto microscope slides. Histological evaluation and immunohistochemistry were performed at the histology core facility at Carver College of Medicine at the University of Iowa. Slides were stained with (i) hematoxylin and eosin (H&E), (ii) CD31 to evaluate the degree of tumor angiogenesis, and (iii) Ki-67 to measure tumor cell proliferation and growth. Stained samples were visualized using a CKX41 Inverted Microscope equipped with a DP70 Digital Camera System (Olympus, Japan). The scale bar was added using ImageJ.

$$\text{Tumor}\text{volume}(\text{m}{\text{m}}^3)=\text{length}\left(\text{mm}\right)\times \text{width}\left(\text{mm}\right)\times \text{height}\left(\text{mm}\right)\times \pi /6$$ Equation 4

In this study, vital organs and blood samples were also collected from euthanized mice to assess the safety of the treatments. On the last day of the in vivo efficacy study (day 50 post-tumor inoculation), 3 mice were randomly selected to study the safety of the treatments. Briefly, mice were anesthetized, and blood was collected via cardiac puncture. Blood samples were incubated for 30 minutes at room temperature, and this was followed by centrifugation for 15 minutes at 16,000 xg and 4°C. Serum samples (supernatants) were harvested to perform various assays to assess hepatic and renal functions, in addition to serum electrolytes, protein levels, glucose levels, and other parameters. Additionally, vital organs (heart, lungs, kidneys, spleen, and liver) from a representative mouse from each study group were collected to evaluate the histology. These organs were washed with 1X DPBS and stored at room temperature in a 10% neutral buffered formalin (Research Products International). Samples were processed onto microscope slides as described above and stained with H&E stains. H&E-stained slides were imaged using a CKX41 Inverted Microscope equipped with a DP70 Digital Camera System. The scale bar was then added using ImageJ.

2.14. In vivo tumor accumulation of CIP2b (soluble vs NPs)

Athymic Nu/Nu mice have challenged SC with 1×10⁶ Hec50co tumor cells per mouse on day zero of the experiment. Once the tumors became palpable, mice were injected IV through the tail vein with either a solution of CIP2b in DPBS:Tween 80:ethanol 85:10:5 (5 mg/kg) or CIP2b-loaded NPs (5 mg/kg) in DPBS (n=3). After pre-determined time points (2, 18, 24, and 36 hours), mice were humanely euthanized using CO₂ followed by cervical dislocation, and the tumors were collected and stored at −80°C. To extract the drug, thawed tumors (on ice) were homogenized, and the drug was extracted as described above in the biodistribution study. The drug extract in ethyl acetate (4 mL) was evaporated under a vacuum, and the residue was reconstituted as described above. Finally, the clear supernatant was injected into the HPLC for CIP2b quantification.

2.15. Safety of CIP2b NPs

BALB/c mice (n = 3 male and 3 female mice) were treated with either PBS (control), CIP2b NPs (5 mg/kg), or blank NPs (equivalent to CIP2b NPs loaded with 5 mg/kg CIP2b) containing no CIP2b. Each mouse received a total of three doses over 1 week, and their body weights were monitored twice a week. On the 14^th day, mice were euthanized, their vital organs were collected and weighed, and blood and sera samples were also collected as previously described. Blood samples were collected to evaluate red blood cell count, hemoglobin, hematocrit, mean globular volume, and mean corpuscular hemoglobin, while sera were used to evaluate hepatic and renal functions, serum proteins, and electrolytes). If a mouse’s body weight declines by more than 20% compared to the initial weight, this mouse will be considered dead. Also, other stress signs including piloerection, curling in the corner of the cage, inability to feed or drink normally, limited mobility, or dehydration, will be monitored.

2.16. Statistical analysis

Data were compared either by two-tailed Student’s t-test or by one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test to compare groups as deemed appropriate. Statistical analysis was performed using Prism 8 (GraphPad Prism, La Jolla, CA). Values where p<0.05 were considered statistically significant.

3. RESULTS AND DISCUSSION

3.1. Physicochemical characterization of CIP2b

Figure 1a shows the chemical structures of CIP2b and CIP. Figure 1b shows the UV-VIS spectra of CIP and CIP2b, as CIP has one UV maximum at 281 nm while CIP2b has two UV maxima at 247 and 281 nm. Subfigures 1c and 1d show the FR-IR spectra of CIP and CIP2b, respectively, while subfigures 1e and 1f show the main signals in the FR-IR spectra of CIP and CIP2b, respectively. Compared to CIP, CIP2b has one extra functional group of C-Cl in the fingerprint region (piperazine group), as shown in the FT-IR spectra. Figure 1g shows the solubility of CIP2b in different solvents/surfactants. The solvent with the highest CIP2b solubility was found to be DMSO, with solubility close to 30 mg/ml. PEG 400, Tween 20, PEG 200, Cremophor EL, and Tween 80 showed relatively higher solubility compared to other solvents and/or surfactants, with values ranging between 2 and 3.5 mg/ml. On the other hand, in aqueous media at various pH values, CIP2b solubility gradually decreased from pH 2 to pH 6, then started to gradually increase from pH 7 and showed a peak at pH 10 (Fig. 1h and 1i). Poor solubility of a drug substance in acidic conditions could be a challenge for oral formulation development as a result of low drug dissolution rate and poor absorption [25]. In this work, however, CIP2b was administered parenterally by IV injection in all in vivo experiments. The experimental intrinsic solubility (log So) was found to be –0.26 (0.5 μg/mL) and this value is lower than the solubility predicted by ChemAxon. Two pKa values were found at 5.4 and 6.0. Also, there was another inflection point near pH 12 which could be another potential pKa.

Figure 1. Physicochemical properties of ciprofloxacin derivative (CIP2b).

(a) Chemical structures of ciprofloxacin (CIP) and its derivative (CIP2b). (b) UV-VIS spectrum shows that CIP has one UV maximum at 281 nm while CIP2b has two UV maxima at 247 and 281 nm. (c) and (d) FT-IR spectra of CIP and CIP2b, respectively. Compared to CIP, CIP2b has one extra functional group of C-Cl in the fingerprint region (piperazine group). (e) and (f) FT-IR frequency range and functional groups of CIP and CIP2b, respectively. (g) Solubility of CIP2b in organic solvents and solubilizers (data are plotted as means ± SD, n=3). (h) Solubility of CIP2b in aqueous buffered solutions at different pH where CIP2b solubility gradually decreased from pH 2 to pH 6, then started to gradually increase from pH 7 and showed a peak at pH 10 (data are plotted as means ± SD, n=3). (i) Solubility (Log S)–pH profile of CIP2b. The experimental intrinsic solubility (log So) was found to be –0.26 (0.5 μg/mL) and this value is lower than the solubility predicted by ChemAxon. Two pKa values were found to be 5.4 and 6.0. There was another inflection point near pH 12 which could be another potential pKa.

3.2. Synergistic effects of PTX and CIP2b combination

Previous work assessing the effectiveness of PTX in treating LOFp53 EC showed chemo-resistance in the Hec50co cell line, which are cells derived from metastatic type II endometrial cancer [10]. To determine if CIP2b synergizes with PTX to alter EC cell growth, CIP2b with PTX was tested against LOFp53 EC and then cytotoxicity was measured. The results demonstrated the poor efficacy of CIP2b or PTX when they were administered individually. However, combined treatments of CIP2b and PTX produced a synergistic effect in a dose-dependent manner that reduced the survival of Hec50co cells (Figures 2a and 2b) while the addition of CIP with PTX showed no benefit over PTX alone (Figure 2c). In addition, the PTX + CIP2b drug combination has been recently tested against three other EC cell lines (Ishikawa-H wild-type p53, Hec50co gain-of-function p53, and KLE gain-of-function p53), and the results demonstrated that the cytotoxic effects were cell line-dependent [14]. For example, incubation of Ishikawa-H wild-type p53 cells with PTX + CIP2b concentrations resulted in marginal cytotoxic activity compared to the effect of the drug combination against the other EC cell lines.

Figure 2. In vitro antitumor activity of combined CIP2b and PTX treatment against Hec50co endometrial cancer cells.

a-c) Cytotoxicity assays of Hec50co cells following treatment for 72 hours with: a) different concentrations of PTX and fixed concentrations of CIP2b (10, 25, and 50 μM) (n=4); b) different concentrations of CIP2b, and fixed concentrations of PTX (1, 5, and 10 nM) (n=4); and c) different concentrations of PTX and a fixed concentration (10 μM) of either CIP2b or CIP (n=4). d) The synergy between CIP2b and PTX is calculated using the Combination Index (CI) method, with a fixed concentration of PTX (5 nM) being added to CIP2b (CI values less than 1 indicate synergy). e) The effect of the addition of 10 μM CIP2b to 1, 5, and 10 nM of PTX for 72 h (n=4). f) The effect of the addition of 5 nM PTX to 1, 10, and 25 μM of CIP2b for 72 h (n=4). g) IC₅₀ values for PTX following the addition of 10, 25, and 50 μM of CIP2b (n=4). h) IC₅₀ values for CIP2b following the addition of 1, 5, and 10 nM of PTX (n=4). i) Distribution of cells in each stage of the cell cycle after receiving the indicated treatment for 24 hours. j) Histograms of Hec50co cells with indicated treatments representing cell cycle phases following the cell cycle analysis using ModFit LT (ver. 5.0.9, Verity Software House) (n=1). Statistical analysis was carried out by one-way ANOVA followed by Tukey’s post-hoc test; *, ***, and **** indicate p<0.05, p<0.001, and p<0.0001, respectively; ns, not significant.

When different concentrations of CIP2b (1, 10, 25, 50, and 100 μM) were combined with 5 nM of PTX, the enhancement of PTX cytotoxicity was found to be synergistic as the calculated Combination Index (CI) values of all combinations tested were <1 (Compusyn^®, Figure 2d). This synergy was further supported by the abrupt drop in relative cell viability from 80 and 95% to <20% upon treatment with 5 nM PTX and 10 μM CIP2b (p<0.001, Figure 2e). Similarly, combining 25 μM CIP2b, with 5 nM PTX reduced cell survival even further to 14% (p<0.001, Figure 2f). The 2.8- to 4-fold reduction in the IC₅₀ values for PTX combined with CIP2b over PTX alone (Figure 2g) and 7- to 18-fold reduction in IC₅₀ values for CIP2b combined with PTX over CIP2b alone (Figure 2h) confirmed this proposed synergism. Cell cycle analysis of Hec50co cells revealed that the addition of CIP2b to PTX increased the percentage of cells in the G2/M phase; specifically, the percentage of cells in the G2/M phase increased from 46% for cells treated with 5 nM PTX alone to 66.1% for cells treated with 5 nM PTX and 10 μM CIP2b (Figure 2i and 2j), whereas CIP2b itself (10 μM) had an unnoticeable effect on the distribution of Hec50co cells in the cycle. Collectively, these studies demonstrate that the combined treatment of PTX and CIP2b decreases Hec50co survival, in part by altering the distribution of cells in the cycle. Our recent studies exploring the key G2/M phase regulators found that the drug combination of PTX and CIP2b activated cdc2 by reducing phosphorylation at Tyr15 [14].

3.3. Properties of CIP2b nanoparticles

In addition to enhancing the accumulation of the drug within tumors, formulating CIP2b into polymeric PLGA NPs also allows delivery of the drug in a clinically feasible formulation ready for intravenous (IV) injection. This is particularly important since CIP2b is a newly developed compound and no formulation has been reported to deliver it in vivo. A third advantage of using NPs to deliver CIP2b is to prolong its circulation time in order to evade the components of the mononuclear phagocyte system [26, 27]. Additionally, PLGA NPs have shown great physical and thermal stability [28]. For these reasons, we set out to prepare polymeric NPs encapsulating CIP2b using PLGA as the polymer, and TPGS as the surfactant. TPGS was used as a surfactant in the NP preparation as it inserts itself into the surface of the PLGA NPs via its lipophilic tocopheryl side while its hydrophilic PEG chains coat the NP outer surface, thus rendering the NPs hydrophilic and therefore capable of evading opsonization and early uptake by macrophages [20, 29–33]. Finally, the slow release of the encapsulated drug at physiological pH ensures that the loaded dose is not prematurely dumped into the plasma before reaching the tumor [27].

The proposed structure of the prepared NPs is illustrated in a schematic diagram (Figure 3a). CIP2b-loaded NPs (CIP2b NPs) were prepared using a nanoprecipitation method. A scanning electron photomicrograph of CIP2b-loaded NPs revealed a spherical structure with a smooth surface (Figure 3b and Supplementary Figure 1). Dynamic light scattering measurements showed that CIP2b-loaded NPs had an average particle diameter (d.) of 151.6 nm with a narrow size distribution (polydispersity index of 0.107), a net surface charge of approximately −29 mV, which helps maintain the stability of the NP in a colloidal suspension [34], and a drug loading of ~881 μg/mL (Figure 3c, 3d, and 3e). In vitro cumulative release of CIP2b from the NPs was slow, reaching ~22% by day 10 of the release assay (Figure 3f). According to the Enhanced Permeation and Retention (EPR) phenomenon, nanocarriers below 200 nm d. have the propensity to be trapped within tumors [35–39], whereas other soluble drugs are not [26].

Figure 3. Development and characterization of CIP2b-loaded NPs.

a) Proposed structure of CIP2b-loaded NPs (CIP2b NPs) with an outer shell composed of PLGA and coated with TPGS as a surfactant. b) Scanning electron photomicrograph showing the shape and morphology of CIP2b-loaded NPs. Scale bar = 100 nm. c) Table showing properties of CIP2b-loaded NPs (n=3). d) Particle size distribution of CIP2b NPs. e) Zeta potential distribution of CIP2b NPs. f) Cumulative in vitro release of CIP2b from NPs in release medium containing PBS and 0.1% w/v Tween-80 for 10 days (n=3). g) Hec50co intracellular accumulation of CIP2b when administered in either soluble form or as CIP2b-loaded NPs (n=3). Statistical analysis was carried out using a two-tailed Student’s t-test (*** indicates p<0.001). h) Quantitation of flow cytometric analysis of the intracellular accumulation of PTX-OG (400 nM) in Hec50co cells in the presence or absence of either CIP2b-loaded NPs (equivalent to 4 μM CIP2b) or blank NPs (equivalent to the amount of CIP2b-loaded NPs used) (n=3). i) Representative flow cytometric histograms of intracellular accumulation of PTX-OG by Hec50co cells. j) Cytotoxicity assay following the treatment of Hec50co cells with different concentrations of PTX, and fixed concentrations of either CIP2b-loaded NPs (equivalent to 0.1 or 0.5 μM of CIP2b) or blank NPs (equivalent to the amount of CIP2b-loaded NPs used) for 72 hours (n=4). k) The effect of adding CIP2b-loaded NPs (equivalent to 0.1 or 0.5 μM CIP2b) or blank NPs (equivalent to the amount of CIP2b-loaded NPs used) to 0.1 nM PTX for 72 hours (n=4). l) Results from a cytotoxicity assay following the treatment of Hec50co cells with indicated concentrations of CIP2b NPs or blank NPs (equivalent to the amount of CIP2b NPs used) for 72 h (n=4). m) Distribution of cells in each stage of the cell cycle after receiving the indicated treatment for 24 hours. n) Cell cycle analysis of Hec50co cells treated with either 10 nM PTX + CIP2b NPs (0.5 μM CIP2b), or 10 nM PTX + blank NPs (equivalent to the amount used for the CIP2b NP treatment). Statistical analyses were performed using either one-way or two-way ANOVA followed by Tukey’s post-hoc test unless noted otherwise. **, ***, and **** indicate p<0.01, p<0.001, and p<0.0001, respectively.

3.4. Comparison between soluble and nanoparticulate CIP2b

CIP2b-loaded NPs were more readily internalized by Hec50co cells in comparison to soluble CIP2b as evidenced by a significant 10-fold increase of CIP2b intracellular accumulation (p<0.0001, Figure 3g). Additionally, Hec50co cells treated with PTX-OG + 4 μM CIP2b-loaded NPs showed a significantly higher accumulation of PTX-OG compared to Hec50co cells treated with PTX-OG alone (p<0.01) or PTX-OG + blank NPs (p= 0.001, Figure 3h and 3i). Results also showed that CIP2b-loaded NPs significantly augmented the cytotoxicity of PTX in comparison to the effect of blank NPs (Figures 3j and 3k). It is worth mentioning that the CIP2b-loaded NPs showed a dose-dependent improvement of cytotoxic effects against Hec50co cells, whereas the corresponding concentrations of blank NPs did not show a similar decline (Figure 3l). Also, when 0.5 μM CIP2b-loaded NPs were combined with 10 nM PTX, the proportion of the cell population in the G₂/M phase was 75.1% as opposed to 67.2% when the combination of PTX + blank NPs was used (Figure 3m and 3n) and 69.5% when 10 nM PTX alone was used (Figure 2j).

3.5. Pharmacokinetics and biodistribution of CIP2b-loaded NPs

Plasma concentrations following the IV injection of CIP2b or CIP2b NPs were monitored for up to 24 h, and the data are represented in Fig. 4a, while Fig. 4b represents the first 300 min of plasma concentration-time profile in Fig 4a. Pharmacokinetic analysis of the data revealed that the NPs were able to prolong the half-life (t_1/2) and the mean residence time (MRT) of CIP2b compared to the free form. In addition, the higher C₀ value in the case of CIP2b NPs implies a marked modification of the tissue distribution profile as will be discussed later. Figures 4d–i display fitted representations of CIP2b levels in different organs (e.g., lung, liver, kidney, heart, spleen, and pancreas), while Fig. 4j shows the area under the curve at 0–24 and 0-infinity either when CIP2b or CIP2b NPs were used. It is clear that free CIP2b had a tendency to accumulate in the lungs of mice, achieving about 4-fold higher AUC 0–24. Even though it is not clear whether this finding is due to the higher tendency of CIP2b itself to distribute to lung tissues possibly as a result of higher CIP2b lung proteins affinity, or due to other reasons. Further experiments are needed to investigate this finding, and in all cases, it is clear that formulating CIP2b into NPs seemed to evade excessive lung accumulation. Injection of CIP2b NPs markedly prolonged the circulation time of CIP2b in the plasma of mice, which in turn increases the chance of tumor accumulation afterward, as will be discussed later. Furthermore, avoiding marked lung accumulation may help administer larger doses of the drug using the NPs without having concerns about possible lung injury due to massive accumulation of the drug there. It is also expected that the actual half-life of unencapsulated CIP2b may be shorter than what is shown in Fig. 4c because the marked lung accumulation possibly provided a reservoir of the drug that gets slowly released into the plasma of mice, falsely prolonging the measured half-life of CIP2b. This was completely avoided when the NPs were used, which enables more accurate and reproducible detection of plasma levels following IV injection.

Figure 4: Pharmacokinetic and biodistribution profiles of CIP2b and CIP2b NPs in mice.

a) Pharmacokinetics following IV injection of CIP2b solution or CIP2b NPs (75 μg/mouse) in female BALB/c mice for up to 24 hours (n=3 mice/time point, data represent mean ± SD). b) close-up view of the pharmacokinetic profile represented in the dashed rectangle in a). c) Pharmacokinetic parameters following non-compartmental analysis (PK Solver) following IV injection of CIP2b solution or CIP2b NPs (75 μg/mouse) in female BALB/c mice for up to 24 hours (n=3 mice/time point, data represent mean ± SD). Biodistribution profiles fitted to 1-compartment model following the IV injection of CIP2b solution or CIP2b NPs (75 μg/mouse) in female BALB/c mice for up to 24 hours (n=3 mice/time point) in d) lungs, e) livers, f) kidneys, g) hearts, h) spleens, and i) pancreas of mice (PK Solver). j) Areas under the curve at 0-t (AUC 0-t) and 0-inf (AUC 0-inf) in lungs, livers, kidneys, hearts, spleens, and pancreas obtained following 1-compartmental fitting of organ concentrations-time profiles represented in d) to i).

There also seems to be a slightly higher initial accumulation (15–20%) of CIP2b in the kidneys and hearts of mice when NPs were used compared to free CIP2b (Fig. 4f and 4g), and further investigation of the drug’s safety in these organs was carried out to make sure the use of NPs does not cause any toxicity in vital organs, as will be revealed later. Furthermore, pancreatic accumulation of CIP2b seems to be low regardless of the dosage form used (Fig. 4i), however, CIP2b NPs seem to provide longer residence in the pancreas compared to the free CIP2b, which may make it potentially attractive to employ CIP2b in pancreatic cancer therapy.

3.6. In vivo antitumor efficacy against human endometrial cancer xenografts

Fig. 5 shows the in vivo antitumor efficacy following IV injection of PBS, PTX + CIP2b, PTX + Blank NPs, and PTX + CIP2b NPs in Hec50co tumor-bearing athymic Nu/Nu mice. IV injection of PTX + CIP2b in tumor-bearing mice marginally reduced tumor volume compared to PBS-treated mice, albeit not significantly (Figure 5a). In contrast, tumor growth was significantly inhibited in mice treated with PTX + CIP2b-loaded NPs (p<0.05, Figure 5a), while PTX + Blank NPs did not show any remarkable tumor inhibition compared to the PBS-treated group. Further optimization of the CIP2b:PTX ratio could improve the outcomes as suggested by the combination index results (Fig. 2d). In the in vivo studies, 1:1 CIP2b:PTX (5 mg/kg dose each) was used due to the limited solubility (0.5 mg/mL) of CIP2b in the vehicle (DPBS:Tween-80:ethanol 85:10:5) [14]. Regardless of the treatment group, there were no differences in body weight among all groups (Figure 5b), suggesting these treatments had low or no toxicity. At the end of the study (day 50 post-tumor challenge), tumors were harvested, imaged, and weighed (Figure 5c). The average tumor weight from mice treated with PTX + CIP2b NPs only was significantly less than the average weight of tumors from the PBS-treated group (Figure 5d). These findings demonstrate that combined treatment of EC tumors with soluble CIP2b and PTX is less effective when delivered systemically via IV injections compared to combined treatment with PTX and CIP2b NPs. Recently, we found that the antitumor efficacy of the CIP2b/PTX combination (each 5 mg/kg) was significantly higher than the negative control (PBS), while PTX alone was not, where treatments were administered peritumorally to Hec50co tumor-bearing athymic Nu/Nu mice [14]. Similar results with different drug formulations are commonly found in the literature [10, 12, 40] and can be directly explained by the enhanced permeation and retention phenomenon (EPR) [35–39]. To confirm that this enhanced efficacy is explained by enhanced tumor accumulation due to the EPR, Hec50co tumor-bearing athymic Nu/Nu mice were injected IV with soluble CIP2b or CIP2b-loaded NPs. Intra-tumoral concentrations of CIP2b were consistently higher at 24 hours following treatment (p<0.05) and did not decline for 36 hours in mice that received CIP2b-loaded NPs versus soluble CIP2b (Figure 5e). In contrast, tumors from mice injected IV with CIP2b solution displayed a steep decline in their CIP2b levels, with significantly lower average intra-tumoral levels at 36 hours compared to intra-tumoral levels at 18 and 24 hours (p<0.01; Figure 5e). This highlights the importance of using NPs with enhanced tumor accumulation capabilities to minimize off-target accumulation of the drugs. Our previous findings also demonstrated that the intra-tumoral concentration of PTX significantly increased upon co-delivery of CIP2b compared to the treatment of PTX alone [14].

Figure 5. Antitumor efficacy of PTX + CIP2b.

a) Hec50co tumor progression in athymic Nu/Nu mice injected IV with indicated treatment (n=6–7). b) Body weight monitoring of the mice following their IV treatment with PBS, PTX + blank NPs, PTX + CIP2b solution, or PTX + CIP2b NPs. c) Photographic image of representative tumors collected from mice treated with IV treatments. d) Average tumor weights following mice euthanasia and tumor collection from mice treated with the previously mentioned IV treatments (n=6–7). e) Intra-tumoral accumulation of CIP2b following the IV injection of either CIP2b solution or CIP2b NPs in Hec50co tumor-bearing athymic Nu/Nu mice (n=3). f) Immunohistochemistry analysis of specimens stained with H&E, or antibodies against CD31 (indicating vascular endothelial cells), or ki-67 (indicating proliferating cells). Yellow arrows point to either the necrotic areas (H&E) or where antibodies showed abundant binding to their specific target. Scale bar = 50 μm. Statistical analyses were carried out by one-way ANOVA followed by Tukey’s post-hoc test or a two-tailed Student’s t-test. * and ** indicate p<0.05 and p<0.01, respectively; ns, not significant.

3.7. Histology and Immunohistochemistry

Compared to other treatment groups, H&E staining of tumors collected from mice treated with PTX + CIP2b-loaded NPs showed broad necrotic areas and an abundance of pyknotic cells with condensed chromatin (Figure 5f), indicating necrosis and/or apoptosis [41]. This demonstrates that combined treatment of PTX + CIP2b-loaded NPs effectively induces cancer cell death inside tumors. Immunohistochemical staining of tumors collected from mice treated with PTX + CIP2b-loaded NPs (versus PBS, PTX alone, or PTX + soluble CIP2b) further supported the efficacy of this treatment combination in inhibiting tumor progression. For example, vascular endothelial cells, as detected by CD31, were scarce in tumors from mice treated with PTX + CIP2b-loaded NPs compared to tumors from mice in the other treatment groups. Moreover, endometrial tumors are usually characterized by a high level of vascularization [10], which is evident by the abundance of vascular endothelial cells (PBS group, Figure 5f) and in contrast to other tumor types, such as pancreatic tumors, which are characterized by necrotic avascular areas in their centers [15]. The absence of visible endothelia in mice that received PTX and CIP2b-loaded NPs suggests that this treatment may have inhibited the neovascularization of tumors. This could be further investigated in future studies aimed at delineating the effect of PTX and CIP2b NPs treatment on vascular endothelial growth factor (VEGF) pathways as a means to combat cancer cells. Staining the tumor tissues with a Ki-67-specific antibody indicated that tumors from mice treated with PTX + CIP2b-loaded NPs possessed fewer proliferating cells compared to other treatment groups (Figure 5f and Supplementary Figure 2).

3.8. Safety of CIP2b and PTX combination

To assess the safety of the combined treatment with PTX and CIP2b-loaded NPs, we euthanized mice on the last day of the experiment (day 50 post-tumor challenge) and harvested vital organs and sera. Vital organs were fixed, embedded, and histologically examined following H&E staining, and several serum parameters were evaluated (e.g., liver functions, electrolytes, and kidney function tests). We did not detect necrosis or visible injury to the tissues (including apoptosis, karyorrhexis, pyknosis, or inflammatory cell infiltration) in any treatment groups (Figure 6a). Additionally, no significant differences in the parameters measured in the serum samples were recorded between treatment groups (Figure 6b). These data, combined with the absence of any significant change in body weight (Figure 5b), suggest that the doses used were safe and absent of any apparent toxicity.

Figure 6. In vivo toxicity profile of CIP2b + PTX combination.

a) Histological evaluation of major vital organs following the IV administration of PBS, PTX + blank NPs, PTX + CIP2b solution, or PTX + CIP2b NPs; H&E staining was performed on sections from the organs collected from representative mice from each group. Scale bar = 50 μm. b) Serum parameters measured in athymic Nu/Nu mice following IV injection of PBS, PTX + blank NPs, PTX + CIP2b solution, or PTX + CIP2b NPs. Statistical analysis was carried out by one-way ANOVA followed by Tuckey’s post-hoc test (n = 3 mice selected randomly from each group, ‘ns’ indicates not significant).

3.9. Safety of CIP2b-loaded NPs

None of the three groups (i.e., CIP2b NPs, blank NPs, or PBS) showed any sign of toxicity following the dosage regimen described above in the experimental section. Neither male nor female mice showed any significant decline in body weight over the span of two weeks (Supplementary Figure 3a). Organ weights were normalized to the body weight of mice, and their values were compared. No significant difference among any of the groups was found (Supplementary Fig. 3b). In addition, RBCs counts and hemoglobin-related value in the CIP2b NPs group did not show any noticeable difference among the groups, indicating no hemolysis (Supplementary Fig. 4). Finally, enzymatic levels, serum protein values, and electrolyte values in the sera of mice showed no significant difference among all the tested groups (in either male or female mice, Supplementary Fig. 5).

4. CONCLUSION

Our work describes the efficacy of using the fluoroquinolone derivative, CIP2b, in combination with paclitaxel to slow tumor progression in animal models of endometrial cancer. This has the potential to replace chemotherapeutic cocktails that involve multiple reagents, which are often deemed necessary for many cancers due to tumor heterogeneity and multidrug resistance. However, the off-target effects of the current strategies can limit their antitumor efficacy and therefore overall effectiveness. In contrast, CIP2b-loaded NPs synergistically augmented the antitumor efficacy of PTX. In vivo evidence indicated a strong safety profile for CIP2b NPs with or without PTX, as none of the treated mice experienced any noticeable toxicity. Notably, IV delivery of CIP2b-loaded NPs significantly increased the tumor accumulation of CIP2b relative to when it was delivered in solution, which likely potentiates the function of PTX at the tumor site. Correspondingly, mice treated with PTX + CIP2b-loaded NPs showed significantly slower tumor growth. This proposed therapeutic strategy, outlined in Fig. 7, has the potential to reduce PTX-related adverse events by enabling the use of lower PTX doses. Further studies will be performed to explore the effect of administering a high CIP2b:PTX ratio and the impact of co-delivering PTX and CIP2b in the nanoparticle-based formulation. This drug combination has the potential capacity to reduce PTX resistance, while simultaneously providing therapeutic antitumor benefits and reducing toxicity. PTX + CIP2b combinatorial therapy may also potentially be used to combat other types of cancers (e.g., breast cancer) that exhibit resistance to PTX monotherapy.

Figure 7.

Graphical representation of the proposed combination therapy to treat endometrial cancer. CIP2b which was found to strongly synergize with PTX against resistant endometrial cancer cells was encapsulated into PEGylated NPs to provide longer circulation and enhanced tumor accumulation. When given with PTX, these NPs provided higher tumor accumulation of CIP2b, which in turn augmented the intracellular cytotoxic activity of PTX. This proposed treatment may lead to the successful reduction of the commonly used PTX doses without compromising its activity.