(T1330-05-27) Co-delivery of Curcumin and Sildenafil Citrate with 3D-Printed Collagen-Chitosan Scaffolds for Enhanced Wound Healing

Purpose: Wound healing remains a significant challenge due to its complex process involving hemostasis, inflammation, proliferation, and remodeling in tissue repair or regeneration[1]. Injuries such as full-thickness skin are associated with high mortality and morbidity, with 500,000 patients treated annually in the United States[2]. Advancements in the therapies for wound healing are needed to alleviate this healthcare concern. Curcumin is a natural hydrophobic compound with anti-infective, anti-inflammatory, antioxidant, and healing properties. Curcumin has proven to be effective against various chronic diseases, including impaired wound healing[3]. Sildenafil citrate is an FDA-approved selective and potent inhibitor of cGMP-specific phosphodiesterase type 5 used as a vascular dilator for treatment of erectile dysfunction and pulmonary hypertension. Nitric oxide (NO) and many messenger molecules also play functional and critical roles in the wound healing process. Studies have shown that sildenafil also promotes wound healing due to its ability to sustain the cGMP-enhancing effect of NO release but has poor physiochemical properties[4]. There is an increasing interest in using 3D printing and additive manufacturing (AM) technologies for creating drug delivery devices with more combinations of biomaterials and drug compounds over the past decade. Both collagen and chitosan are biodegradable and biocompatible polymers used clinically as drug delivery matrices. Our objective was to design and fabricate 3D-printed collagen-chitosan scaffolds with curcumin and sildenafil citrate to aid in wound healing.

Methods: ollagen 2% and chitosan 3% (w/v) powder were dissolved in 1% acetic acid solution and stirred at low temperature. Curcumin and sildenafil citrate were added to the uniform hydrogel. The bioinks were printed using a BioX extrusion-based 3D bioprinter from Cellink (San Carlos, CA) and crosslinked with UV exposure for 12 hours. Scaffolds were stored at -80 °C for 24 hours then lyophilized for 24 hours. The rheological behavior of the gels was measured using an MCR-302 Anton Paar (Graz, Austria) rheometer. The rheology was measured at 25°C, with an increasing shear rate from 0.07 to 100 s^-1. Surface morphology and roughness of the scaffolds were determined with a Keyence VHX-6000 digital microscope (Itasca, IL) at 200× and 500× magnifications. Water retention capacity was measured by comparing the weight of the scaffold after immersing in 20 mL PBS incubated at 37°C and the initial scaffold weight. Thermal analysis was performed by differential scanning calorimetry (TA Instruments Model Q200, New Castle, DE), from 10 to 300°C at 10°C /min. Individual specimens were weighed (6–7 mg) using sealed aluminum pans. In vitro drug release studies were carried out in PBS: ethanol (8:2 v/v) at 37℃ over one week. Media was removed and replaced with fresh release media at each sampling point and analyzed by high-pressure liquid chromatography (HPLC) with UV detector. The cumulative release percentage was plotted as a function of time. All data were represented as mean±standard error of the mean (n=6).

Results: Figure 1 illustrates the printing process of collagen-chitosan scaffolds. The 3D printer with a temperature controller and a syringe extrusion printhead was used for printing. Table 1 shows the optimized printing parameters such as nozzle diameter, extrusion rate, printing speed, print bed temperature, and infill density that need to be utilized to obtain scaffolds with good printability. Surface morphology of 3D-printed scaffolds is shown in Figure 2A with 200× and 500× magnification. The images (Figure 2A (c&d)) of drug loaded scaffolds showed uniform drug deposition and distribution in the formulations. The rheological properties (Figure 2B (a)) present a trend that viscosity of bioinks decreases sharply with a shear rate of 10 s^-1. Drug-loaded hydrogel displayed greater viscosity than the polymer hydrogel with the addition of drug. Figure 2B (c) shows the DSC curves of pure drugs, polymers, and printed scaffolds. Sildenafil citrate and curcumin showed endothermic (melting point) peaks at 197 and 184°C. The glass transition temperature for collagen and chitosan was observed at 92°C. The physical mixture curve depicts the melting peak of drugs and polymers similar to their pure forms, suggesting the crystalline nature of the drug. A broad endothermic peak of both scaffolds’ curves at 99 and 97°C for non-drug loading and drug loading scaffolds, respectively. Besides, both drug profiles (Figure 2C) showed a burst release in the initial 12 hours followed by a slow-release rate within 7 days. Almost all the sildenafil citrate (~97%) was released, and around 60% of curcumin was eluted over time.

Conclusion: 3D printed co-drug-loaded scaffolds were prepared using syringe extrusion-based 3D printing technology. The DSC curves for 3D-printed scaffolds suggest thermal stability of collagen-chitosan scaffolds, and the potential to decrease the degradation speed of later release behavior. Reduction of roughness for drug-loaded scaffolds can increase the biocompatibility of formulations. A novel analytical HPLC method was developed successfully to simultaneously quantify the co-drug, saving sample preparation time and improving efficiency. This technique allows for personalized wound healing applications.

References: 1. Akbik, D., et al., Curcumin as a wound healing agent. Life Sci, 2014. 116(1): p. 1-7.
2. Padula, W.V. and B.A. Delarmente, The national cost of hospital-acquired pressure injuries in the United States. Int Wound J, 2019. 16(3): p. 634-640.
3. Kumari, A., et al., Wound-Healing Effects of Curcumin and Its Nanoformulations: A Comprehensive Review. Pharmaceutics, 2022. 14(11).
4. Septifani, E.A., Y.R. D., and A. R., Review: The Discovery and Development of Sildenafil Citrate. Asian Journal of Pharmaceutical Research and Development, 2021. 9(4): p. 108-117.


Figure 1. The schematic illustration of (A) Bioinks preparation. (B) 3D-printed collagen-chitosan scaffolds fabrication.


Table 1. Optimized 3D printing parameters for the fabrication of collagen-chitosan scaffolds.



Figure 2. (A) Surface morphology of 3D printed scaffolds. Top view of the scaffolds of (a) (b) Collagen-chitosan, (c) (d) Curcumin and sildenafil citrate loaded in collagen-chitosan (200×, 500× magnification). (B) (a) Rheological properties of the bioinks with shear rate at room temperature. (b) Swelling determination of printed scaffolds with various drug loaded percentage at 37°C. (c) DSC thermograms of pure sildenafil citrate, curcumin, collagen-chitosan, physical mixture, and 3D-printed scaffolds. (C) In vitro drug release profile of 3D-printed scaffolds loaded with 5% (w/w) sildenafil citrate and 0.5% (w/w) curcumin at 37°C for 7 days.