Abstract
Colorectal cancer (CRC) is the 3rd most lethal type of cancer-linked deaths worldwide. Combination chemotherapy offers a better therapeutic response against CRC than single drug therapy. The purpose of this study was to successfully encapsulate Capecitabine (CAP) and Regorafenib (REG) in liquid crystal nanoparticles or cubosomes (CUBs) matrix for targeting CRC following intravenous (IV) administration. Active targeting of the CUBs was achieved by surface capped hyaluronic acid (HA) owing to its binding affinity with complex differentiation (CD-44) receptors on cancer stem cells. The HA-capped REG-CAP co-loaded CUBs (HA-REG-CAP-CUBs) were prepared via the Top-bottom method and optimized using Design Expert®. The optimized formulation had a mean particle size of 196.1 ± 1.4 nm, polydispersity index (PDI) of 0.231 ± 0.03, zeta potential (ZP) of -25.5 ± 5.2 mV and entrapment efficiency (%EE) of REG and CAP was 78.56 ± 2.1% and 76.48 ± 2.5%, respectively. Morphological studies revealed uniform distribution and smooth cubic texture of the CUBs. Solid state characterizations suggested no interactions among the ingredients of the prepared system. The HA-REG-CAP-CUBs showed a significantly reduced HCT116 (4.39 ± 1.30%) and H29 (2.39 ± 0.53%) cell viability in 48 h. The pharmacokinetics study revealed a controlled release profile of CAP and REG with excellent biodistribution and prolonged plasma circulation over 48 h, thereby leading to 20.3 ~ and 10.2 ~ fold improved bioavailability, respectively. Histopathological studies demonstrated the safety of the HA-REG-CAP-CUBs towards vital organs in an animal model. It is concluded that HA capped CUBs may be suitable carriers for the targeted delivery of combinational antitumor drugs.
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Introduction
Globally, CRC is the 3rd most diagnosed and 2nd most common cause of cancer mortality. During the past three decades, the CRC cases have increased by 157%1. In a recent study, the CRC early-onset incidence rate was highest among countries like Australia, United States, New Zealand and South Korea in different age groups2. The major contributing factors for CRC among people below 50 years include obesity, smoking, alcoholism, socio-economic status, unhygienic dietary patterns and sedentary lifestyle3. High human development index (HDI) countries are more vulnerable to CRC than low-to-medium HDI countries. Owing to genomic instability (either chromosomal or microsatellite), mutations in tumor protein 53 (TP53), adenomatous polyposis coli (APC), Kirsten rat sarcoma (KRAS) and mismatch repair (MMR) genes4 facilitate an uncontrolled segregation of gut glandular epithelial cells. This change results in the development of an adenomatous polyp, which, if left untreated, could form malignant CRC over 10–15 years, as shown in Supplementary Figure S15. The development of CRC from early-stage (adenomas and polyps) to late-stage tumors (malignant) is a complicated and intricate process, affected by complex molecular signaling pathways. Therefore, various strategies are adopted to combat CRC, including radiation therapy in early stages, chemotherapy, immunotherapy and combination therapies6.
Chemotherapy is considered an optimal strategy for cancer management to target abnormally growing cells and prevent relapses, most importantly, the combination of chemotherapeutic agents has recently been applauded in CRC treatment. Regorafenib (REG) was approved as an oral chemotherapeutic agent by the Food and Drug Administration (FDA) in 2012 for combating CRC. It works by blocking EGFR, PDGFR, FGFR, VEGFR, KIT, RET, TIE-2, CSF-1R and receptors tyrosine kinase (RTKs). As a result, it prevents angiogenesis in tumor micro-environment (TME), blocks the blood flow towards CRC, and provides antitumor immunogenicity via tumor associated macrophages (TAMs) modulation7. Various clinical trials, like randomized double-blind placebo-controlled Phase III trial CORRECT (NCT01103323) and the CONCUR trial (NCT01584830), have proved the therapeutic efficacy of REG against CRC. REG is also recommended for the treatment of hepatocellular carcinoma (HCC), gastro-intestinal stromal tumor (GIST). However, REG being a member of the Biopharmaceutics Classification System (BCS-2), is associated with poor solubility and dissolution profile and may pose adverse effects like hepatotoxicity, diarrhea, mucositis and hypertension8,9. Capecitabine (CAP), a prodrug of 5-Fluorouracil (5-FU), belongs to the 1 st line chemotherapy against CRC that gets converted into 5-FU by thymidine phosphorylase (TP) in the TME. It blocks the thymidine synthase (TS) enzyme needed for thymidine nucleotide synthesis, and the absence of thymidine stops the DNA replication10. The elimination half-life is approximately 1 h, which compromises the therapeutic efficacy in CRC. CAP is approved for the treatment of breast, pancreatic, and gastric cancers. The reported adverse effects of CAP include cardiotoxicity, myelosuppression, gastro-intestinal disturbance and hand-foot syndrome. CAP is linked with extensive metabolism, low bioavailability, systemic toxicity owing to rapid metabolism, less plasma/systemic circulation and non-specific targeting, which undermines its therapeutic efficacy11. Various carriers have been used for the delivery of chemotherapeutic agents, such as conventional and advanced drug delivery carriers, to produce desired therapeutic effects.
Nanomedicine-based advanced drug delivery carriers have tremendous applications in chemotherapeutic drug delivery. Several nanoparticles, including liposomes, polymeric micelles, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), dendrimers, metallic nanoparticles, polymeric nanoparticles (PMs) and multifunctional liquid crystal nanoparticles (LCNPs) have been used in pre-clinical and clinical studies for targeted delivery against CRC12,13. Cubosomes (CUBs) are self-assembled, amphiphilic, multi-dimensional lyotropic nanoparticles with high porosity, better biocompatibility and %EE of hydrophilic, amphiphilic and lipophilic drugs. They offer controlled release, improved bioavailability, and enhanced stability14. The internal structure could be primitive, diamond or gyroid type, with a bi-continuous cubic phase consisting of two non-communicating aqueous channels running across a single continuous bilayer membrane15. Hyaluronic acid (HA) ligand-based surface conjugation enables CUBs with specific binding ability to complex differentiation (CD44) receptors, which are over-expressed on cancer stem cells, thus allowing active targeting and tumor-only cell specific uptake, leading to apoptosis16,17. Similarly, a biocompatible lipid like glyceryl mono-oleate (GMO; HLB value: 1–3) improves the efficiency of CUBs owing to its non-toxic, bio-compatible, and generally recognized as safe (GRAS) characteristics18.
The rational of this study was the development of HA-REG-CAP-CUBs with controlled release, improved bioavailability, efficacy in tumor cell lines and lower systemic toxicity towards the treatment of CRC. The formulation was statistically optimized using Design Expert®, followed by investigating its particle properties, surface morphology, solid state characterization, including Fourier transform infra-red (FTIR) spectroscopy, powder x-ray diffractometry (PXRD) and proton nuclear magnetic resonance (1HNMR) analyses. Moreover, in vitro release and anticancer studies in HCT-116 and H29 cell lines were accomplished to observe the release profile and antitumor potential of the HA-REG-CAP-CUBs, respectively. Additionally, pharmacokinetic experiments were performed, followed by histopathological analysis of the major organs of Albino Wistar rats to check bioavailability and safety of the prepared formulation. To the best of our knowledge, no such study of HA-capped CUBs, incorporating REG and CAP, has so far been reported for the treatment of CRC. The potential mechanism of action of the developed HA-REG-CAP-CUBs for the treatment of CRC is described in Supplementary Figure S2.
Materials and methods
Chemicals
REG was a kind gift from the College of Pharmacy & Pharmaceutical Sciences, Hanyang University (Ansan, South Korea). CAP, Dimethyl sulfoxide (DMSO), and HA (MW 12–14 kDa) were bought from Maklin, manufactured by Shanghai Chemical Industry Park, Shanghai, China. Tween 80 and Stearyl amine (SA) were bought from Sigma Aldrich (Germany). GMO (99% purity) was obtained from Zhengzhou Jiuyi Time New Materials Co., Ltd. (Zhengzhou, Henan, China) through a local vendor. Organic solvents (Ethanol, Chloroform) and deionized water were obtained from local vendors (Islamabad, Pakistan). Human colon cancer cell lines (HCT116 and HT29 cells), methyl-thiazolyl-tetrazolium (MTT) assay kit, were bought from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). H&E staining was performed at Ali Labs, Islamabad, Pakistan. All other materials used were of analytical grade.
Animal study
Albino Wistar rats (weight 200 ± 25 g and age 8–10 weeks) were brought from the National Institute of Health (NIH), Islamabad, Pakistan. They were kept in the animal house with a standard temperature of 24–25 °C and relative humidity, i.e., 50 to 60%. Isoflurane (3–5% in 100% oxygen) was given via inhalation using a precision vaporizer system at a flow rate of 1–2 L/min to induce anesthesia. The animal studies were performed in accordance with the relevant guidelines and regulations, including the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (Version 2), the American Veterinary Medical Association (AVMA) guidelines for the Euthanasia of Animals (2020) and the NIH animal welfare act and regulations. Additionally, the experimental protocol of this study was approved by the bioethical committee of Quaid-i-Azam University, Islamabad, Pakistan, with approval no. BEC-FBS QAU2024-582.
Preparation of HA-SA conjugate
The coating amphiphile HA-conjugate SA was formulated by carrying out an amide reaction as reported earlier19. Momentarily, HA (100 mg), along with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide EDC (100 mg) and N-hydroxy succinimide NHS (60 mg) were dissolved in deionized water (10 mL) under constant stirring for 8 h. The temperature of the system was maintained at 60 °C, and the magnetic hotplate was set at 600 rpm. The process was carried out to activate the carboxylic acid (-COOH) functional group of HA, which later forms a conjugation (covalent bond) with the amine group (-NH2) of the SA. Simultaneously, in a glass vial, SA was weighed (71 mg) and then dissolved in N, N-dimethylformamide (DMF): Chloroform (3:1) mixture. Then the SA solution was dropwise added into the HA mixture with gentle stirring (600 rpm at 60 °C for 6–12 h). The reaction was kept stirring for another 24 h. Then, the reaction product undergoes dialysis using a dialysis membrane with an external medium consisting of water: ethanol (1/1, 1/2, 1/3 v/v) for the next 48 h. After that, the reaction product was centrifuged (Hermle labortechnik, Z 216 MK, Germany) at 12,000 rpm for 1 h. The supernatant was then removed, and the precipitates were collected and dried via lyophilization, i-e., freeze drying (8–12 h, at − 80 to − 40 °C). The dried product was stored in an airtight Eppendorf tube for future analysis such as FTIR, solubility profile testing and 1HNMR.
Method of preparation of CUBs
Blank CUBs were prepared with necessary modifications in the method reported by Al-Mahallawi et al.20 and shown in Supplementary Figures S3 and S4. Briefly, 150 mg GMO, 30 mg HA-SA conjugate, 30 mg Tween 80, and 1mL chloroform were taken together in a glass vial as the organic phase and heated at 60 °C on a hotplate until the molten state was formed. In another vial, 10 mL of deionized water was heated to the same temperature. After that, the molten organic phase was added dropwise to the aqueous phase under 700 rpm mechanical stirring. Afterwards, the mixture was shifted to bath sonication for one min to get a homogenous milky white mixture, which was stored for 1 h in the fridge (2–8 °C). Then, it was subjected to probe sonication having 40% amplitude, 3 s ON and 3 s OFF cycle for 40 s. The finalized blank CUBs dispersion was stored in a fridge for stability and was later investigated for optimization. REG (5 mg) and CAP (10 mg) loaded CUBs were prepared following the same method, where both drugs were taken in the organic phase along with 3 mL ethanol for drug dissolution and later, evaporated during molten state formation.
Optimization of HA-REG-CAP-CUBs via box-behnken design (BBD)
To achieve desired critical quality attributes (CQAs), Design Expert® software (Version 12; Stat-Ease, Minneapolis, MN, USA) was used for optimization of HA-REG-CAP-CUBs. Briefly, the influence of the independent variables like concentration of the GMO (X1), Tween 80 (X2), and CAP (X3) was checked for dependent variables like particle size, PDI, ZP, and %EE of drugs using 33 factorial BB factorial study design (Supplementary Table S1, S2, S3)21. Analysis of variance (ANOVA) statistical tool was applied to verify robustness of data, and p value below 0.05 was considered statistically significant.
Characterization of HA-REG-CAP-CUBs
Evaluation of size, PDI and ZP
The mean particle size distribution, PDI and ZP of HA-REG-CAP-CUBs were evaluated using Zeta Sizer ZS 90 (Malvern Instruments, Worcestershire, UK), supplied with Helium and Neon laser working at 635 nm wavelength. All measurements were taken in triplicate (mean ± S.D.) and done at 90° fixed light incidence angle and temperature 25 °C using zeta sizer software version 6.34. Before the investigation, 10 µL of the sample was diluted with 1 mL deionized water, followed by vortexing for one min. Afterwards, the CUBs were evaluated for their appearance and content homogeneity22.
Evaluation of %EE
%EE of HA-REG-CAP-CUBs was determined by calculating the concentration of free drug in the supernatant. Briefly, 2 mL of HA-REG-CAP-CUBs was centrifuged at 13,500 rpm for 2 h at 4° C21. Supernatant was collected and examined by a UV-visible spectrophotometer at 260 nm8 and 307 nm23 for REG and CAP one after the other in the same sample. %EE of the HA-REG-CAP-CUBs was measured by the given formula
Cryogenic transmission electron microscopy (cryo-TEM) analysis
For the detailed structural configuration of HA capped CUBs, cryo-TEM (JEOL JEM-1400Plus, Tokyo, Japan) was used. Briefly, a droplet of CUBs formulation was placed onto hydrophilized (using glow discharge) lacey carbon-coated Cu grid. The Cu grids were kept at 22 °C with saturated humidity to minimize evaporation during sample preparation and analysis. Then, the sample was frozen to −183.15 °C in a controlled environment following rapid immersion into liquid propane/ethane and liquid nitrogen for vitrification. After the removal of excess ethane/propane using filter paper, the specimen was transferred to the cryo-TEM holder operated at 200 kV under bright mode. Afterwards, the zero-loss filtered scans were recorded at low dose conditions digitally using a camera24.
Scanning electron microscopy (SEM) analysis
SEM analysis of the HA-REG-CAP-CUBs was performed to evaluate morphological characteristics. The SEM (SSX-550, Shimadzu, Kyoto, Japan) was used to capture the images of HA-REG-CAP-CUBs at a potential of 5 to 20 kV. The formulation was first dried overnight via lyophilization, and the dried formulation was then fixed onto a glass slide supported by double sticky tape so that it could be secured onto aluminium stubs. The samples were then coated/covered with carbon tape under an argon atmosphere. Using a high energy electronic beam, which interacted with the sample, surface morphology and size of HA-REG-CAP-CUBs were revealed after completion of sample scanning, and the results were recorded afterwards25.
FTIR analysis
FTIR analysis was carried out to check the possible physicochemical interactions among the components of HA-REG-CAP-CUBs. For this purpose, samples of pure CAP, pure REG, HA, SA, GMO, Tween 80, and lyophilized HA-REG-CAP-CUBs were analyzed using dry crystalline potassium bromide (KBr) in a 1:100 ratio. For this, the sample and KBr were compressed into disks for the transmittance readings. All spectra were captured using an FTIR spectrometer (OMINIC™ software spectra, PerkinElmer spectrum RX FTIR, USA) from 4000 to 500 cm− 126.
PXRD analysis
PXRD investigations were conducted using pure REG, CAP and HA-REG-CAP-CUBs to analyze the crystalline nature. Many diffractograms were obtained from XRD (model AXS D8 Advance, Bruker, USA), which were attached with a copper anode (Cu Kα radiation, λ = 1.5406 Å) linked with a Si (Li) position sensitive detector (PSD). The current (I) and the voltage were both set at 100 mA and 40 kV, respectively. The speed of the scan was set to 0.03 s, and the diffracted intensities of the samples were recorded. The DIFFRAC plus software was used to record the diffraction patterns. The data was generated in scan mode with a step size of 0.02° (2θ) and a 2θ scan range of 10–80°27.
1HNMR spectrum analysis of HA-SA conjugate
The newly synthesized HA-SA conjugate was characterized by 1HNMR, using a 400 MHz apparatus (AVANCE 500, Bruker, Germany), to determine the successful conjugation between the HA and SA and to evaluate the environment of hydrogen atoms (H-atoms) present both in the SA and HA. Moreover, the behavior of each H-atom with respect to the neighbor H-atom was investigated. The equipment was run at 400 MHz with 500 µL solvent (deuterated chloroform (CDCl3), having 15 mg of HA-SA product dissolved in it. The NMR tube with the dissolved sample was covered to prevent the evaporation of the solvent, fluctuation in the concentration-volume and the qualitative peak results were analyzed accordingly25,28.
In vitro release studies
In vitro release analysis was performed using the dialysis bag method in different dissolution media, including pH (5.5, 6.8, and 7.4) to mimic the TME and the normal physiological blood pH, respectively29. Analysis was performed in a 250 mL beaker that had 200 mL phosphate buffer saline (PBS) of the respective pH. The dialysis membrane was divided into equal pieces, and one end was tied using a clip. An equivalent dose of 5 mg REG dispersion and 10 mg CAP dispersion was added separately and tied on the other hand as well. The membrane tubes were at once immersed in the beakers. Before the addition of formulations, the dialysis membrane was soaked in the respective buffer solution for 30 min. The system’s temperature was adjusted to 37 ± 0.5 ˚C. The speed of the shaking bath was kept at 100 rpm. At time intervals of 0.5, 1, 2, 4, 6, 8, 12, and 24 h, a 2 mL aliquot from each formulation was withdrawn and analyzed using LC-MS/MS for drug quantification. An equal volume of buffer was added to the system to keep the sink condition. The % cumulative drug released from the test formulations was plotted against the respective time (h). The release kinetics of REG-dispersion, CAP-dispersion and the HA-REG-CAP-CUBs were figured out by plotting the data against the zero order, Korsmeyer-Peppas, first order, Higuchi, and Hixson-Crowell release models using DD solver software30.
Cell viability studies
The cytotoxic potential of the HA-REG-CAP-CUBs was investigated using the MTT assay, and the results were compared with the REG-CAP-dispersion, REG-dispersion, CAP-dispersion, blank CUBs and normal saline (0.9% NaCl)31. The samples were prepared by making separate serial dilutions of the mentioned stock solutions in a 96-well plate. Two different CRC cell lines, mainly HCT-116 and H29 cells, were used to carry out cell viability studies. Briefly, 10 × 10³ HCT116 and H29 cells per well were seeded in a 96-well plate, which was then kept in an incubator at 37 °C. Then, the cells were subjected to different concentrations (1.56–100 µg/mL) of HA-REG-CAP-CUBs, REG-CAP-dispersion, REG-dispersion, and CAP-dispersion. The final volume of each well in the plate was 200 µL, and the 96-well plate was incubated for 24 h and 48 h periods32. Afterwards, the cell culture was treated with MTT (0.5 mg/mL; 20 µL) and PBS for 2–4 h under incubation. The reaction resulted in the production of Formazan due to the reduction of the MTT. The medium was then replaced by DMSO to dissolve formazan crystals after 4 h, and samples were seen by a microplate reader at the wavelength scan of 570 nm. The experiment was conducted in triplicate.
Stability studies
Stability studies of the prepared HA-REG-CAP-CUBs were performed, according to the International Council on Harmonization (ICH) guidelines, for a 3-month period at 4 °C, 25 °C and accelerated temperature of 40 °C. For this purpose, HA-REG-CAP-CUBs were stored at different temperature conditions, and samples were collected at 0, 1-, 2-, and 3-month intervals to check possible changes in the size, PDI, and %EE of drugs33.
Pharmacokinetic evaluation
Pharmacokinetic study of the HA-REG-CAP-CUBs was investigated in albino Wistar rats to observe its bioavailability and compare it with the REG- and CAP- Dispersion. The rats were divided into three groups, each having 5 rats (n = 5). The first group was given a volume of 0.4 mL HA-REG-CAP-CUBs intravenously (IV) via tail vein (containing 5- and 10-mg/kg REG and CAP, respectively). One of the remaining two groups was given REG Dispersion (5 mg/kg)34 and the other was given CAP Dispersion (10 mg/kg)35,36. Soon after the dose administration (5 min), a 250 µL plasma (blood) was withdrawn via the tail vein. The remaining samples were taken at 30 min, 1 h, 2, 4, 6, 12, 24 and 48 h, centrifuged at 9000 rpm for 10–15 min using a centrifuge (HERMLE, Germany). Plasma samples were then separated and stored at −20 °C before the plasma drug concentration (Cp) determination. At a suitable time, plasma (150 µL) was assorted with 250 µL of acetonitrile solution containing 100 µL of 10 µg/mL of paclitaxel as an internal standard. It was then centrifuged at 9000 xg for 10 min to precipitate the proteins. The supernatant (10 µL) was inserted into the Liquid Chromatography Mass spectrometer (LC-MS) for quantification of REG and CAP. The standard curve and chromatograms of plasma, regorafenib and capecitabine are displayed as Supplementary Figures S5 and S6, respectively. The liquid chromatography system used consisted of Rheos Allegro quaternary pumps, equipped with an online degasser and an HTS PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) controlled by Janeiro-CNS 1.1 software (Flux Instruments AG, Thermo Fisher Scientific Inc., Waltham, MA, USA). The chromatographic separation was performed on a 2.1 mm × 75 mm X select™ HSST3–3.5 μm analytical column (Waters, Milford, MA, USA) placed in a temperature-controlled column heater (HotDog 5090, ProLab GmbH, Reinach, Switzerland). The chromatography system was coupled to a triple-stage quadrupole (TSQ) quantum mass spectrometer (MS) from Thermo Fischer Scientific, equipped with an electrospray ionization (ESI) interface and operated with the Xcalibur 2.0.7 software (Thermo Fischer Scientific Inc., Waltham, MA, USA)37.
LC-MS/MS conditions
Chromatographic separation was done with a mobile phase composed of 2 mM ammonium acetate in ultrapure water with 0.1% formic acid (FA) (solution A) and acetonitrile with 0.1% FA (solution B). The mobile phase was delivered at a flow rate of 0.3 mL/min, using a stepwise gradient elution program detailed in Table S4. The temperature-controlled column heater was set at 25 °C, and the autosampler was kept at 10 °C. The optimum potential settings and MS/MS transitions were chosen during direct infusion into the MS/MS detector of each analyte solution separately at a concentration of 0.1 µg/mL in methanol: water (MeOH/H2O) 1:1. The selected m/z transitions and the collision energy (CE) were 483.1 m/z for REG, 487.1 m/z CAP and 270.1 m/z for Paclitaxel; internal standard (IS) respectively. Scan time and scan width were 0.02 s and 0.5 m/z, respectively, and each chromatographic peak was the result of ca. 30 scans. The capillary temperature for ionization was set at 350 °C. The ESI spray voltage was set at 3.8 kV, and the source-induced dissociation was kept at 10 V. The sheath and auxiliary gas (nitrogen) flow rates were set at 60 and 10 (arbitrary units), respectively. The Q2 collision gas (argon) pressure was 1 mm Torr. Chromatographic data acquisition, peak integration and quantification were performed using the Xcalibur LC Quan software package. The mass spectrometer was run with the ESI source Ion Max in positive mode. Samples were analyzed via selected reaction monitoring (SRM) detection mode, employing the transition of the [M + H] + precursor ions to product ions38,39. Analytes were detected by electrospray triple-stage quadrupole mass spectrometry and quantified using the calibration curves with stable isotope-labeled internal standards. The method was confirmed based on FDA recommendations, including assessment of extraction yield (74–104%), matrix effects, and analytical recovery (94–104%) with low variability (< 15%). The method was sensitive (lower limits of quantification within 250 ng/mL or 0.25 µg/mL), accurate (intra- and inter-assay bias: −0.3% to + 12.7%, and − 3.2% to + 6.3%, respectively) and precise (intra- and inter-assay CVs within 0.7–7.3% and 2.5–8.0%, respectively) over the clinically relevant concentration range (upper limits of quantification 250 to 25,000 ng/mL or 25 µg/mL).
Pharmacokinetics data & statistical analysis
Chromatographic data acquisition, non-compartmental evaluation, peak integration and quantification were performed using the Xcalibur LC Quan software package. The level of significance of the data was obtained using analysis of variance (ANOVA) tests. All the data were presented as the mean ± standard deviation (S.D.) or as the mean (medians) for Tmax, and the data were considered as significant at a level p ˂ 0.0540,41.
Histopathological analysis
After the collection of the last samples for pharmacokinetic studies, the animals were euthanized. Euthanization was performed in a non-pre-charged CO2 chamber following the 2020 AVMA Guidelines. The CO2 flow was regulated with 30–70% of the chamber volume per minute, maintained for > 60 s following respiratory arrest (which may take up to 5 min). Moreover, euthanasia was assured by cervical dislocation and the visceral organs of test animals, particularly their liver, kidney and heart, were excised, washed with normal saline 2–3 times and stored in 50 mL of freshly prepared 10% Formalin buffer. The organs were then rinsed with distilled water, followed by cleansing with xylene, dilute ethanolic solution and covering with paraffin wax for block preparation. Then, 5 μm of the respective tissue was cut and mounted on the glass slides after de-paraffinizing, re-hydrating and rinsing using xylene/ethanolic solution. The slides were submerged in hematoxylin and eosin (H&E staining kit) reagents for approximately 10–15 min, followed by rinsing with PBS (pH 7.4). The slides were subjected to another cycle of drying, washing and were finally covered with a cover slip. A compound microscope was used at 10X magnification for the investigation of the respective tissues architecture42.
Statistical interpretation
In the current study, all experiments were performed in triplicate, and the data has been represented as mean ± standard deviation (mean ± S.D.). Design Expert software (V-12), with statistical tools, one way ANOVA, was used for the optimization of CUBs. Sigma plot (V-12.5), PK solver and GraphPad Prism (V-9) were used for the interpretation of results and generating the required graphs. The data showing p value < 0.05, < 0.01, and < 0.001 were considered statistically significant43.
Results
Preparation of CUBs
The amphiphilic HA-SA conjugate was synthesized for surface modifications of CUBs. The FTIR and 1HNMR characterizations confirmed it. Likewise, the HA-REG-CAP-CUBs were successfully prepared using Top-bottom approach with probe sonication for homogeneity. The clear milky white cubosomal formulation had uniform distribution with no phase separation upon storage.
Optimization of the HA-REG-CAP-CUBs
Blank CUBs prepared via the Top-bottom approach expressed a mean size of 132.8 ± 5.3 nm, PDI of 0.197 ± 0.06, and ZP of −19.3 ± 0.42 mV as given in Table S2. Un-capped CUBs expressed 180.2 ± 2.32 nm size, PDI (0.183 ± 0.01), ZP (−15.3 ± 1.40 mV), %EE of REG (72.45 ± 2.1%) and %EE of CAP (76.22 ± 1.4%) reported in Supplementary Table (S3). The Box Behnken model via the Design Expert suggested 15 runs for the optimization of HA-REG-CAP-CUBs (Table 1). All the formulations were prepared, and the impact of the independent variables was observed to be significant on the size, PDI, ZP, and %EE of REG and CAP of HA-REG-CAP-CUBs. The size was in the range of 156.4 to 258.1 nm, PDI was found in between 0.147 and 0.326, and the ZP in between − 38.1 to −9.78 mV. Moreover, the %EE of REG was observed to be between 58.36 and 82.47% and the %EE of CAP between 59.08 and 84.69%. Formulation 9 was selected as the optimized formulation with particle size (196.1 ± 1.4 nm), PDI (0.231 ± 0.03), and ZP (−25.5 ± 5.2 mV) as shown in Fig. 1. The %EE of REG and CAP was respectively found to be 78.56 ± 2.1% and 76.48 ± 2.5% in HA-REG-CAP-CUBs (p < 0.05). The GMO facilitated entrapment of both the drugs in the CUBs, such that the lipophilic REG was incorporated in the lamellar bilayer and the amphiphilic CAP was entrapped in the aqueous core and along the water channels running across the cubic phase. Increased GMO concentration showed an increase in size and PDI, a more negative ZP and an increase in %EE of lipophilic REG and CAP. Moreover, the concentration of Tween 80 (stabilizer) significantly influenced the dependent variables. Such that, increased Tween 80 concentration significantly decreases the particle size, ZP, PDI and %EE of the drugs. Similarly, augmented CAP concentration showed a slight increase in particle size, a decreased negative ZP, and a marginal increase in PDI. Additionally, it decreased the %EE of REG, and increased the %EE of CAP. The three-dimensional (3D) graphs highlighted the impact of independent variables on the dependent variables. The graphs representing the effects of variables on the size, PDI and ZP, %EE are shown in Fig. 2.
Cubosome characterization (A) particle size and PDI; (B) zeta potential of HA-REG-CAP-CUBs.
3-D Graphical presentation of the parameters affecting (A) particle size; (B) zeta potential; (C) PDI; (D)%EE; (REG); (E)%EE (CAP). (i) Effect of GMO and Tween 80, (ii) Effect of Tween 80 and CAP (iii) Effect of GMO and CAP. The graphs were plotted using Box Behnken Design of Design Expert® software.
Morphological characterization of HA-REG-CAP-CUBs
Cryo-TEM analysis
The HA-REG-CAP-CUBs were seen utilizing cryo-TEM for morphological characteristics. The results showed that the CUBs were monodispersed, uniformly distributed with no aggregation Fig. 3(A). The sample expressed CUBs have interconnected water channels (ultra-internal structure). The cubic geometry was slightly masked by the surface modification of CUBs using HA. The cryo-TEM images showed that prepared CUBs have a proximity close to the particle size characterization via dynamic light scattering (DLS) i-e. ˂ 200 nm.
Morphological characteristics of the optimized HA-REG-CAP-CUB using (A). cryogenic transmission electron microscopy (Cryo-TEM) and (B). scanning electron microscopy (SEM).
SEM analysis
SEM was used for visualization of the surface morphology and topography of the HA-REG-CAP-CUBs. It revealed that the prepared CUBs agglomerates were spherical, with smooth texture appearance and a lack of characteristic roughness when observed at magnification (30,000X) as shown in Fig. 3 B.
Solid state characterization
FTIR analysis
The FTIR results of the HA-REG-CAP-CUBs and their components are displayed in Fig. 4. The spectrum of the lipid GMO showed many peaks in the region of 4000 to 500 cm− 1. Mainly, the peaks were at 692 cm− 1, 1190 cm− 1, 2850 to 2950 cm− 1, and 3200 cm− 1 to 3500 cm− 1, revealing bond stretching for the -CH2 and -OH functional moieties. The FTIR of Tween 80 expressed various peaks at 1100 cm− 1, 2800 to 2900 cm− 1, and 3200 to 3500 cm− 1, respectively for -C-O, -CH2 bond bending and -OH group stretching. The FTIR spectrum of CAP showed peaks highlighting respective functional groups at 1080 cm− 1, 1120 cm− 1, 1500 cm− 1, 1620 cm− 1, and 2950 cm− 1 for -C-O, -C-F, -N = O, -C = C, and -CHO groups. For REG, the spectrum highlighted peaks with respect to functional groups at 650 cm− 1, 1050 cm− 1, 1120 cm− 1, and 3500 cm− 1, respectively, for -C-Cl, -C-O, -C-F, and -NH groups. The FTIR spectrum of HA revealed characteristic peaks at 1050 cm− 1, 1600 cm− 1, and 3400 cm− 1 for -C-O, -C = O, and -OH functional groups, respectively. The SA expressed several peaks at 690 cm− 1, 1200 cm− 1, 1740 cm− 1, between 2850 cm− 1 and 2980 cm− 1 for -CH, -NH, and -CH2 groups, respectively. The FTIR spectra of HA-SA conjugate displayed various peaks at 690 cm− 1, 1080 cm− 1, 1560 cm− 1, 1650 cm− 1, and 2900 cm− 1 for -CH, -C-O, -C = O, -C-N, and -CH2 group, respectively. The HA-REG-CAP-CUBs expressed characteristic peaks at 650 cm− 1, 1080 cm− 1, 1500 cm− 1, 1560 cm− 1, 1650 cm− 1, between 2800 cm− 1 and 2900 cm− 1, bond stretching between 3300 cm− 1 and 3400 cm− 1 and bending at 3500 cm− 1 for -C-Cl, -C-O, -N = O, -C = O, -C-N, -OH and -NH groups, respectively.
Fourier Transform Infrared (FTIR) analysis of the optimized HA-REG-CAP-CUB and its components, including GMO, Tween 80, REG, CAP, HA, SA, and SA-HA.
FTIR of the HA-SA conjugate
The HA-SA conjugate was successfully synthesized via covalent amide functional bond formation between the -COOH (carboxylic acid) of HA and the -NH2 (amine) group of the SA. The characteristic peaks at 1650 cm− 1 (amide 1 -C = O in acylamide) and 1560 cm− 1 (-C-N amide 2 in acylamide) revealed the synthesis of HA-SA conjugate as shown in Supplementary Figure S7.
PXRD analysis
PXRD analysis of the CAP and REG revealed their crystalline nature by indicating the characteristic crystalline peaks. The XRD spectrum of the CAP revealed various crystallinity peaks at 10.10°, 12.21°, 17.95°, 20.2°, 28.32°, 42.12°, while the XRD spectrum of the REG expressed characteristic peaks at 14.21°, 15.11°, 20.24°, 25.52°, 33.26°, 37.23°, indicating their crystallinity in pure form (Fig. 5). However, the absence of major peaks in the XRD spectrum of HA-REG-CAP-CUBs highlighted that the drugs changed their crystalline nature during the process of CUBs formation, thereby converting to an amorphous form. The XRD spectrum showed that both drugs were successfully entrapped in the CUBs after being solubilized in their respective phases, and changed their crystalline nature into an amorphous. This change may be vital in improving the solubility of both drugs, which may improve their bioavailability.
Powder X-ray Diffraction analysis (PXRD) of HA-REG-CAP-CUB, pure CAP, and pure REG.
1HNMR spectrum of the HA-SA conjugate
The 1HNMR spectrum of the synthesized HA-SA conjugate revealed characteristic peaks of H-atoms in both HA and SA with respect to the neighboring H-atoms. The spectrum revealed several peaks for the HA-SA conjugate (Fig. 6). Briefly, the peaks at 0.97, 1.27 and 2.99 ppm expressed the methyl (-CH2) group of the SA. The peaks ranging between 3.30 ppm and 3.48 ppm revealed H-atoms of the HA-sugars (glycosidic H-atoms). The characteristic peak of HA-SA at 1.77 ppm was attributed to the -NHCOCH3 group.
Proton nuclear magnetic resonance (1HNMR) spectrum of the HA-SA conjugate.
In vitro release studies
The release profile of HA-REG-CAP-CUBs was compared with REG-dispersion and CAP-dispersion at all the evaluated pH. The pKa value of REG lies between 10.5 and 12.0 as reported in the literature and it shows a release profile according to the pH of the external environment. The REG-dispersion gave maximum release at pH 7.4 and minimum in acidic conditions. As can be seen at pH 7.4, REG-dispersion released around 64.34 ± 2.1% REG after 4 h, followed by 96.38 ± 1.4% at pH 7.4 after 8 h (Fig. 7Ai). Similarly, at pH 6.8, the REG Dispersion released 54.42 ± 0.94% REG after 4 h and around 79.83 ± 1.92% after 8 h (Fig. 7Aii). Additionally, at pH 5.5, around 53.51 ± 1.9% and 74.93 ± 1.4% REG was released at 4 h and 8 h, respectively (Fig. 7Aiii). The REG-dispersion showed a release profile up to 8 h and followed 1 st order release kinetics. However, in the case of HA-REG-CAP-CUBs, the release was hindered by surface modification with HA at pH 7.4. Overall, approximately 50.89 ± 1.81%, 73.39 ± 2.03% and 82.40 ± 2.39%, REG was respectively released from HA-REG-CAP-CUBs at pH 7.4, 6.8 and 5.5 after 24 h, as shown in Fig. 7Ai-iii.
CAP is a prodrug with a pKa value of 8.8, its release profile was investigated at all three pH. At pH 7.4, CAP-dispersion showed 68.13 ± 2.1% and 99.04 ± 2.1% release in 2 h and 4 h, respectively (Fig. 7Bi). At 6.8 pH, it demonstrated a corresponding CAP release of 56.03 ± 2.03% and 74.85 ± 2.59% after 4 h and 8 h (Fig. 7Bii). Moreover, at 5.5 pH, a further diminished release of CAP was observed at 4 h (44.28 ± 1.75% and 8 h (60.39 ± 2.13%) (Fig. 7Biii). The CAP-dispersion showed a release profile following 1 st order release kinetics. The HA-REG-CAP-CUBs showed a pH dependent release profile of CAP, such that the release was maximum at lower pH. As can be seen, around 30.59 ± 0.91% CAP was released from the HA-REG-CAP-CUBs at pH 7.4 after 4 h, followed by 61.38 ± 2.20% release in 24 h. At pH 6.8, the CAP release was found to be 40.89 ± 1.13% after 4 h, followed by 81.48 ± 2.42% after 24 h. Likewise, at pH 5.5, the CAP release was observed to be 49.91 ± 1.83% and 93.39 ± 2.63% after 4 h and 24 h (statistically significant; p < 0.05), respectively (Fig. 7B). The release of CAP and REG from HA-REG-CAP-CUBs followed Korsemeyer-Peppas release kinetics, showing a sustained and controlled release from CUBs during release studies. Supplementary Table S4 and S5 show the release kinetics coefficients and Supplementary Figure S8 shows a graph of release kinetics.
In vitro release profile of REG (A) and CAP (B) at different pH (i; pH 7.4, ii; pH 6.8, iii; pH 5.5) from HA-REG-CAP-CUB and respective drug dispersion. Analysis was performed in triplicate, mean ± SD (n = 3).
In vitro cell lines analyses
In vitro anticancer potential of the HA-REG-CAP-CUBs was evaluated in HCT-116 and H29 cells and compared to normal saline, blank CUBs, REG-dispersion, CAP-dispersion, and REG-CAP-dispersion. The cell viability of normal saline (100.02 ± 3.1% vs. 98.33 ± 4.04%) and blank CUBs (100.04 ± 2.4% vs. 96.38 ± 1.1%) treated cell lines were not meaningfully changed at all the concentrations used. Similarly, their IC50 values were observed to be 49,113 µg/mL and 7240 µg/mL, respectively. However, a significantly reduced cell viability of HCT116 and H29 cells was detected, when treated with HA-REG-CAP-CUBs and REG-CAP-dispersion, at both the observed time periods (24 h and 48 h of incubation) as shown in Fig. 8 (A&B). Similarly, REG-dispersion and CAP-dispersion also expressed decrease in the cell viability of HCT116 (Fig. 8A) and H29 cells (Fig. 8B), at all the tested concentrations. Notably, the HA-REG-CAP-CUBs showed a significantly reduced HCT116 cells viability (15.23 ± 3.39%) in 24 h (Fig. 8AI), followed by further reduced cell viability (4.39 ± 1.30%) in 48 h (Fig. 8AII). Similarly, the REG-CAP-dispersion (23.23 ± 2.21% vs. 17.10 ± 1.22%), REG- dispersion (47.39 ± 3.93% vs. 39.90 ± 3.91%) and CAP-dispersion (38.40 ± 3.28% Vs 29.11 ± 3.46%) also reduced the HCT116 cells viability, even if it was more than the HA-REG-CAP-CUBs, after 24 (Fig. 8BI) and 48 h (Fig. 8BII) of analysis, respectively. In case of the H29 cells, the cell viability was decreased in the manner of HA-REG-CAP-CUBs (13.98 ± 2.36% vs. 2.39 ± 0.53%), REG-CAP-dispersion (22.77 ± 3.29% vs. 12.36 ± 2.32%), REG- dispersion (48.56 ± 3.28% Vs 38.23 ± 3.33%) and CAP-dispersion (40.21 ± 3.67% Vs 30.32 ± 2.23%), respectively in 24 h and 48 h analysis. The IC50 values were found significantly reduced by the HA-REG-CAP-CUBs (3.340 µg/mL) as compared to the IC50 values of REG-CAP-dispersion (7.318 µg/mL), REG-dispersion (50.11 µg/mL) and CAP-dispersion (24.09 µg/mL) after 48 h of treatment of HCT116 cells. Similarly, the IC50 values of HA-REG-CAP-CUBs, REG-CAP-dispersion, REG-dispersion, and CAP-dispersion were observed respectively as 1.39 µg/mL, 4.292 µg/mL, 29.39 µg/mL and 68.39 µg/mL after 48 h of treatment of H29 cells (results were statistically significant, p < 0.05) as shown in Table 2.
In vitro cell line studies of HA-REG-CAP-CUB and its comparison with REG-CAP-dispersion, REG-dispersion, CAP-dispersion, blank cubosomes and normal saline in (A) HCT 116 cells and (B) H29 cells (I) 24 h, 48 h (II). Analysis was performed in triplicate, mean ± SD (n = 3).
Stability analysis
CUBs are considered as thermodynamically stable drug carriers as temperature has little effect on the CQAs of CUBs44. The HA-REG-CAP-CUBs showed non-significant changes in the size, PDI, ZP and %EE of drugs over time. Momentarily, after 3 months of analysis, the mean size was found 205.3 ± 2.1 nm, PDI was 0.235 ± 0.02, and ZP was of −28.8 ± 2.4 mV at 4 °C. Moreover, the size, PDI and ZP were respectively found as 216.2 ± 2.3 nm, 0.254 ± 0.02, and − 33.9 ± 3.9 mV at 25 °C, with slight increase in attributes, however still in the acceptable range. Additionally, the size, PDI and ZP was respectively observed to be 234.7 ± 1.5 nm, 0.256 ± 0.03, and − 45.6 ± 2.8 mV at 40 °C after 3 months (p < 0.05). The detailed results are shown in Table 3.
Pharmacokinetic investigations
Plasma concentration vs. time curve of the HA-REG-CAP-CUBs is displayed in Fig. 9 and the data was compared with REG-dispersion, and CAP-dispersion after IV administration. A significantly enhanced bioavailability of the REG and CAP was observed in HA-REG-CAP-CUBs as compared to their dispersions. Particularly, the AUC (781.21 ± 39.6 ng/mL*h) of HA-REG-CAP-CUBs was meaningfully enhanced as compared to the AUC of REG-dispersion (76.1 ± 21.2 ng/mL*h) and CAP-dispersion (38.4 ± 15.3 ng/mL*h). Moreover, the Cmax of all the testing groups was not significantly different (Table 4). Nevertheless, the HA-REG-CAP-CUBs showed a gradual decrease in the plasma concentration of samples over time. Additionally, the half-life (t1/2) of HA-REG-CAP-CUBs (22.25 ± 2.2 h) was significantly prolonged as compared to the half-life (t1/2) of REG-dispersion (1.20 ± 0.7 h) and CAP-dispersion (0.42 ± 0.09 h). Furthermore, a reduced elimination rat constant (Kel (h− 1)) was observed for HA-REG-CAP-CUBs (0.0287 ± 0.001 h− 1), when compared with REG-dispersion (0.5321 ± 0.002 h− 1) and CAP-dispersion (1.5287 ± 0.003 h− 1), respectively, indicating a sustained release. Overall, the pharmacokinetics study exhibited a meaningfully improved bioavailability of the REG and CAP with improved retention time and broad AUC. More specifically, a 10.2 ~ and 20.3 ~ fold increase in bioavailability of REG and CAP was respectively observed in HA-REG-CAP-CUBs when compared with drug dispersions.
Plasma drug concentration vs. time profile of HA-REG-CAP-CUBs, REG-dispersion, CAP-dispersion in rats. Analysis was performed in quintuplicate, mean ± SD (n = 5).
Histopathological analysis
Histopathological studies revealed that REG-dispersion and CAP-dispersion caused severe tissue damage in the major organs analyses compared to HA-REG-CAP-CUBs. In case of REG-dispersion, histopathological analysis of the liver tissue revealed degenerative hepatocytes marked by nucleus shrinkage, vacuolated cytoplasm, sinusoidal endothelial rupturing (shown by black arrows) around the central vein (CV) (Fig. 10 AI). The kidney showed hemorrhage and necrosis of glomerulus and presence of blood in the collecting duct (CD) (Fig. 10 AII). The cardiac muscle expressed micro-vesiculation of cardiomyocytes, necrosis and Zenker’s degeneration (Fig. 10 AIII). Conversely, the HA-REG-CAP-CUBs didn’t cause any damage to the visceral organs. The liver and kidney tissues expressed normal structural arrangements as shown in (Fig. 10 BI & BII). Similarly, the heart tissue expressed normal configuration of cardiomyocytes (Fig. 10 BIII), thereby showing safety towards the cardiac muscles. However, REG-dispersion resulted in cytotoxicity of the hepatocytes (Fig. 10 CI). The kidney tissue analysis revealed a mild hemorrhage in glomerulus and distal/proximal convoluted tubules (Fig. 10C1I), while the cardiac tissue demonstrated mild damage (pointed by arrows) (Fig. 10 CIII). Overall, the results showed that HA-REG-CAP-CUBs were safe towards the major organs.
Histopathological studies of visceral organs of (A) REG-dispersion, (B) HA-REG-CAP-CUBs and (C) CAP-dispersion. (I) Liver tissue, (II) Kidney tissue, (III) Heart tissue. The images were studied at 10X. The bar length is 30 μm.
Discussion
The HA-capped REG-CAP co-loaded CUBs (HA-REG-CAP-CUBs) aim to unify multi kinase inhibition with antimetabolite cytotoxicity, while overcoming solubility, stability, and scheduling hurdles. Co-loaded CAP and REG cubosomes tackle REG’s poor solubility and CAP’s stability challenges by leveraging bicontinuous lipid nanochannels for dual localization and controlled co-release. These systems are industrially scalable via HPH/microfluidization and dry state stabilization, align with quality by design (QbD) controls, and map onto existing clinical use of REG and CAP combinations. CUBs have been extensively used as drug delivery system for cancer targeting45. Herein, HA-capped CUBs were developed via Top-bottom approach46. HA ligand-based surface conjugation of CUBs was done to enable them to target CD44 receptors, which are over expressed on cancer stem cells14,47. Hyaluronic acid (HA) is a linear polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. The –COOH group comes from the glucuronic acid moiety. The activation of the carboxyl (-COOH) group of HA is usually done to enable chemical conjugation or crosslinking48. The reasons include facilitating covalent bond formation, improving functionalization, crosslinking for stability, and enhancing drug delivery potential49,50.
The concentrations of GMO, Tween 80 and CAP were adjusted, and choice of the optimized formulation was based solely on the CQAs of HA-REG-CAP-CUBs that was best fit for IV administration. Mean particle size, PDI, ZP and %EE of the prepared CUBs were evaluated to obtain the most suitable formulation. It is worth mentioning that %EE was checked at 4 °C to minimize drug leakage. It has been reported earlier that at lower temperatures, lipid bilayer fluidity decreases, reducing the chance of drug diffusion out of the CUBs matrix51. Moreover, many protocols adopt 4 °C as a benchmark condition for %EE to allow reproducibility and comparison across formulations. It’s a trade-off between structural fidelity and analytical stability52.
GMO being amphiphilic lipid in nature can encapsulate hydrophilic, lipophilic as well as amphiphilic drugs. A change in GMO concentration imparted significant increase in the size, ZP and %EE of CUBs21,53. Increasing GMO concentration resulted in a rise in the %EE of REG, most probably due to enhanced cubic phase viscosity with prompt solidification, thereby preventing lipophilic drug diffusion into external medium53. The PDI of CUBs also increased with GMO concentration54. Similarly, ZP of CUBs became more negative with increased GMO concentration, owing to the carboxyl and hydroxyl groups present in GMO structure55,56. Likewise, Tween 80 was used as stabilizer to enhance stability and durability of the CUBs. Increased Tween 80 concentration reduced the size of CUBs, because of the decreased interfacial tension between the cubic and the aqueous phase, thereby preventing aggregation57. Moreover, a decrease in the %EE of CAP occurred due to the solubilization of CAP in aqueous phase and the ZP became less negative. The 3rd variable i-e., CAP concentration also influenced the CQAs of HA-REG-CAP-CUBs. Meanwhile, a marginal increase in the size and ZP of the CUBs was marked by increasing the concentration of CAP. CAP has both hydrophilic58 and lipophilic59 properties and it has the tendency to get entrapped either in the CUBs bilayer or in the water channels running across the cubic phase for drug release60. Run 9 was selected as the optimized formulation with excellent size, suitable PDI, optimum ZP, and notable %EE of REG and CAP. The PDI value below 0.3 expressed that the formulation had uniform distribution of the CUBs61 and the particles were not aggregated in the external medium. The ZP values highlighted an optimum charge on particles to avoid coalescence and aggregation. Generally, nanoparticles with more than ± 15 mV ZP value, have been considered stable62. Ideally, the size for IV administration is below 200 nm25, and the cut-off tumor pore size was reported to be 380–780 nm63 to prevent phagocytosis by RES and rapid elimination, thereby enhancing plasma circulation period of the nanoparticles64,65.
The functional groups of HA-REG-CAP-CUBs and its ingredients were analyzed, and detection of the respective peaks indicated the absence of any chemical reaction. The FTIR peaks of GMO ranged between 2950 and 2850 cm− 1 and 690 cm− 1, 1742 cm− 1 and 3200–3400 cm− 1 for -CH2, -C = O, -OH, respectively were following the reported data66. The REG spectrum peaks at 3388 cm− 1, and 650 cm− 1 for -NH, and -C-Cl functional groups respectively were close to the reported literature67. Similarly, FTIR peaks of CAP were 2859 cm− 1, 1606 cm− 1, and 1206 cm− 1 for -CHO, -C = C and -C-F groups, respectively68. HA-REG-CAP-CUBs expressed the characteristic peaks of its ingredients at specified areas, with no new bond’s formation. FTIR spectrum of HA-SA expressed peaks at 1560 cm− 1 and 1650 cm− 1 showing that the conjugate was successfully synthesized69,70. PXRD analysis revealed the disappearance of the characteristic crystallinity peaks of drugs, indicating a phase shift of drugs from crystalline to amorphous form thereby improving the solubilization and bioavailability of drugs. The reported peaks of crystalline CAP at 5.01°, 10.45°, 18.85°, 20.1°, 25.65°, 28.35°71,72 and REG at 12.84°, 15.01°, 22.23°, 26.05° were in accordance with the reported literature73. The 1HNMR spectrum of the HA-SA conjugate highlighted spectrum of H-atoms considering the neighboring environment. Both groups in the HA and in the SA were detected in the final HA-SA conjugate. The peaks at 0.97 and 1.27 ppm highlighted the presence of -CH219,69. Peak at 1.77 ppm was due to NHCOCH3, whereas the peaks between 3.30 and 3.48 depicted the glycosidic H-atoms of the HA. The emergence of a new peak at 1.2 ppm further verified the successful conjugation of the HA and SA, as reported earlier74. A singlet at 8 ppm gave a sign about amide bond between the -COOH and -NH2. The downfield at 1.7 ppm gave the indication of downfield effect on -CH2 alpha to amine of SA after amide bond formation due to high inductive effect of oxygen attached to -NH2. Cryo-TEM revealed ultrastructure showing that the HA-REG-CAP-CUBs kept observable cubic consistency irrespective of the HA capping and were uniformly distributed throughout the formulation75. SEM image expressed that CUBs were spherically aggregated after drying of the aqueous phase and had smooth surface texture76.
It has been reported earlier that TME has an acidic environment (pH 5.5–6.8) due to abundance in lactate77. Therefore, in vitro release studies were carried out at different pH including pH 5.5, 6.8 and 7.4. The HA-REG-CAP-CUBs portrayed maximum release in the acidic environment, possibly due to the ionization of the drugs in the media. At physiological pH 7.4, the drug release was hindered by surface modification of CUBs. At pH 6.8 and pH 5.5 it was found increased, which was in alliance with the reported results78. Also, HA promoted drug release in the acidic buffer79. The release of CAP from CAP dispersion was decreased as the pH of media was decreased from pH 7.4 to pH 6.8. This could be because of poor ionization of CAP at a lower pH, thereby reducing its release. At 5.5 pH, the CAP release was further diminished80. REG pKa values lie between 10.5 and 12.0 as reported in the literature81. The REG-dispersion showed quick release at pH 7.4, followed by retarded release with a reduced pH level. Comparatively, the HA-REG-CAP-CUB exhibited improved REG and CAP release when the pH was lowered to 5.5. The capping of HA significantly impacted the release behavior of drugs82, both at physiological as well as in acidic pH83. The release kinetic analysis showed that the REG and CAP release from HA-REG-CAP-CUBs followed the Korsemeyer-Peppas model, which is known for expressing anomalous drug release behavior from lipid-based nanocarriers84. The n value (˂ 0.45) suggested that the drugs followed a Fickian diffusion release pattern from the CUBs. Whereas the CAP- and REG- dispersions followed the 1 st order release kinetics model at respective pH.
The HA-REG-CAP-CUBs expressed significantly reduced cell viability of both the HCT 116 and H 29 cells among the tested groups in 24 h and 48 h of analysis, followed by REG-CAP-dispersion, CAP-dispersion and REG-dispersion. This could be attributed to the synergistic action of drugs encapsulated in CUBs, which are promising nanocarriers for controlled release anticancer drug delivery owing to their improved targeting ability, pharmacokinetic profile, safety, and minimal toxicity85,86. Moreover, the CUBs were made of biodegradable/biocompatible components. Additionally, the CAP and REG co-loaded in CUBs showed significantly improved synergistic antitumor effects compared to the individual drug dispersions.
The stability study of HA-REG-CAP-CUBs revealed that the formulation was intact in terms of appearance, uniformity, and distribution. There was a slight change in the size, PDI, ZP and %EE, however, it was not meaningfully different87. The stability of the formulation could be attributed to the preparation procedure, the suitability of the ingredients and the thermodynamically stable nature of CUBs. Moreover, CUBs offer better and long-lasting stability to the entrapped drugs and the overall cubic system. This could be due to the cubic crystallographic symmetry through the self-organization of GMO into bilayers around bi-continuous non-intersecting water channels88. Owing to the higher lipid ratio in the bilayer membrane and stronger electrical repulsion, the CUBs have greater membrane stability than the liposomes while keeping a better inherent physical/chemical stability profile against enzymatic degradation of active pharmaceuticals and offer high %EE of therapeutics60. Additionally, the use of Tween 80 as stabilizer (surfactant) made the CUBs more stable. The stability studies were performed for up to 3 months and need to be extended to at least 6 months.
Results of the pharmacokinetic studies revealed significantly improved bioavailability of REG (10.2-fold) and CAP (20.3-fold) from the HA-REG-CAP-CUBs compared to the drug dispersions. The AUC of the optimized formulation was higher compared to the AUC of both the CAP and REG dispersion, showing that the maximum quantity of drugs was available over the extended period40. In the case of REG-dispersion, there was a rapid decline in the graph, showing that REG was distributed among tissues, metabolized, and then excreted after 6 h, as reported. Likewise, the CAP-dispersion showed a prompt declining graph owing to its rapid absorption and biodistribution after IV administration, high aqueous solubility, and 1st pass metabolism and was excreted in the form of metabolites35. As reported in the literature, CUBs possessed characteristic structural features which allowed controlled release of both the drugs after IV administration89. The results showed that intravenous delivery via cubosomes can be beneficial for the drugs than the commercial oral dosage forms, which undergo hepatic degradation and require larger doses to produce a therapeutic effect.
Histopathological studies revealed noticeable tissue damage in animal groups treated with REG- and CAP- dispersions, as both underwent hepatic metabolism and caused significant hepatic tissue damage90. The normal architecture of hepatocytes converted into degenerative hepatic cells with REG-dispersion, as reported in the literature91. The cardiac tissue was damaged with CAP-dispersion after IV administration. Histopathological studies revealed interstitial edema, hemorrhage, and endothelial hypertrophy92. Unlikely, HA-REG-CAP-CUBs formulation didn’t cause any damage to the vital organs, including the liver, kidney and heart. The HA-REG-CAP-CUBs group expressed intact cellular architecture and uniformity. The tissue parenchyma and vasculature (central vein) supported their integrity, as reported in the case of individual drugs93.
Overall, these results demonstrated an anticipated enhancement in the properties of REG and CAP, their synergy through synchronized cytotoxic, sustained release, improved pharmacokinetics and better tolerability. However, this study does have some limitations, including a lack of in vivo antitumor analysis and detailed practical translational pathways from lab to clinic. Therefore, the author’s future perspective is the testing of HA-REG-CAP-CUBs in an in vivo CRC model using different test animals. Moreover, a detailed toxicity profile of the prepared formulation and explanation of the involved molecular pathways would be investigated in animal models and would be published separately.
Conclusions
The HA-REG-CAP-CUBs were successfully prepared and optimized. The HA-SA conjugate was designed for surface capping of CUBs to achieve active targeting. The HA-REG-CAP-CUBs manifested suitable carrier properties for IV administration. The in vitro release studies showed a controlled release profile, where maximum release was seen in an acidic environment. The formulation was found to be stable during stability studies. The in vitro cancer cell lines studies revealed HA-REG-CAP-CUBs with the highest cytotoxicity towards HCT 116 and H29 cells. Similarly, the pharmacokinetic studies showed a significantly enhanced bioavailability of REG and CAP from the HA-REG-CAP-CUBs with a controlled release profile and exceptional plasma circulation over 48 h. Histopathological studies revealed nontoxic manifestations in the major organs. It was concluded that the CUBs have the potential to sustain the drug release, target the tumor cells and improve the bioavailability of the drugs. However, in vivo testing of the prepared formulation in animal models is highly recommended and our research group is working on it. The results will be published separately upon completion.
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
The authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through Large Research Project under grant number RGP2/567/46. Moreover, the authors are thankful to Prof. Han Gon Choi and Prof. Dr. Ihsan-ul-Haq for their continuous support.
Funding
This study was funded by the Higher Education Commission through its project number (20-14604/NRPU/R&D/HEC/2021).
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M. K.R, S.B. and S.W. Analyze and investigate the data, and wrote the first draftA.H.A. and A.A.F. provides the resources, software and analyze the dataZ.A.A., N.K.A. and M.U.R. helped in methodology, Investigation, and writing & review the manuscriptF.U.D. Supervise and funded the study and helped in project administration.
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Rehman, M.K.u., Batool, S., Woo, S. et al. Hyaluronic acid capped cubosomes co-loaded with antitumor agents towards the treatment of colorectal cancer. Sci Rep 16, 2254 (2026). https://doi.org/10.1038/s41598-025-32089-5
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DOI: https://doi.org/10.1038/s41598-025-32089-5
