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Highly Porous Silk Fibroin Scaffold Packed in PEGDA/Sucrose Microneedles for Controllable Transdermal Drug Delivery Ya Gao, Mengmeng Hou, Ruihao Yang, Lei Zhang, Zhigang Xu, Yuejun Kang, and Peng Xue Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01715 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on February 1, 2019

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Highly Porous Silk Fibroin Scaffold Packed in PEGDA/Sucrose Microneedles for Controllable Transdermal Drug Delivery Ya Gao†,‡ , Mengmeng Hou†,‡, Ruihao Yang†,‡, Lei Zhang§, Zhigang Xu†,‡, Yuejun Kang*,†,‡ and Peng Xue *,†,‡

†

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University),

Ministry of Education, School of Materials and Energy, Southwest University, Chongqing 400715, China. ‡Chongqing

Engineering Research Center for Micro-Nano Biomedical Materials and Devices,

Chongqing 400715, China. § State

Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716,

China. KEYWORDS: Microneedles; Silk fibroin; PEGDA; Sucrose; Controlled drug delivery.

ACS Paragon Plus Environment

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ABSTRACT

Polymeric microneedles have attracted increasing attention as a minimally invasive platform for delivering drugs or vaccines in a more patient-friendly manner. However, traditional microfabrication techniques using negative molds with needle-shaped cavities usually require cumbersome centrifugation and vacuum degassing processes, which have restricted the scaled-up mass production of polymeric microneedles. Herein, a novel polydimethylsiloxane (PDMS)-based negative mold with cavities packed with silk fibroin scaffold is developed for rapid fabrication of polymeric microneedles, which comprise primarily the composition of poly(ethylene glycol) diacrylate (PEGDA) and sucrose as the needle matrix. Fibroin scaffolds can instantly adsorb prepolymer solution due to capillary force, and subsequently initiate the formation of microneedles via photoinduced polymerization. Based on three types of model drugs including Rhodamine B (RhB), indocyanine green (ICG) and doxorubicin (DOX), the fabricated PEGDA/sucrose microneedles can realize effective transdermal delivery and controllable release of therapeutic molecules by regulating the sucrose content. The presented method provides a simple strategy for quick fabrication of polymeric microneedles towards transdermal drug delivery applications.


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1. INTRODUCTION Traditional routes for delivering therapeutic drugs, large biomolecules (peptides, antibodies and nucleic acids) in particular, usually suffer from many intrinsic deficiencies that restrict their therapeutic efficacy in the body.1-3 There is a realistic dilemma in that high dosage of drugs may cause harsh side effects, while low dosage of drugs may result in insufficient bioavailability of therapeutics in the lesions. For instance, drug introduction via oral administration are usually accompanied with premature degradation issues due to the harsh environment in gastrointestinal tract and first pass metabolism in the liver.4, 5 Hypodermic injection of drugs requires professional medical aid, induces severe pains and further produces biohazardous sharp wastes.6,

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Alternatively, transdermal route of drug delivery has a unique advantage similar to oral administration in terms of convenience and patient compliance, while it also avoids the issues of premature elimination of active pharmaceutical ingredients.8, 9 However, the outmost layer of skin, stratum corneum, is a critical barrier and poses a major challenge for passive diffusion of pharmaceutical agents with high molecular weight into the circulatory system.10 An emerging technique to break through the skin barrier is the utilization of microneedles patches, which contain an array of ultra-small sharp projections in the scale of micrometers for minimally invasive drug delivery.11-13 Different from hypodermic needles, microneedles create microconduits in the epidermis layer, allowing drug diffusion from the needles into interstitial fluids and further blood capillaries.14 Attributed to the micron-size structure, microneedles avoid stimulating the end-nerves associated with the feel of pain, which is particularly attractive to the patients with needlephobia.15 The microneedles have been demonstrated to transport various therapeutic agents, from small molecules to nano-/micro-particles, into the dermal or epidermis layers.16, 17 Manufacturing microneedle devices is cost-effective based on well-established and


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scalable micro-molding techniques. For example, microneedles have been produced from a wide variety of material matrices, including metal, silicon and biocompatible polymers for delivery of insulin, hormones and vaccines.18-20 However, metal and silicon microneedles eventually become sharp and biohazardous waste after uses, which poses serious immunogenic risks and environmental issues.18, 19 In contrast, microneedles made of biocompatible polymers enable drug loading in the needle matrix and spontaneous drug release after skin penetration via polymer swelling and dissolution, followed by degradation and excretion from physiological environments.20-24 There are many techniques developed for fabrication of polymeric microneedles, such as micromolding, droplet-born air blowing, drawing lithography, photolithography, solvent casting, continuous liquid interface production and dipping.25-31 Amongst these existing strategies, micromolding is probably the most widely used method attributed to the cost effectiveness, reproducibility and scalable production.25 Particularly, PDMS has been reported as a superior substrate for manufacturing micro-molds with low cost, mechanical flexibility and good thermal stability.32,

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However, many technical challenges still exist in molding of polymeric

microneedles, such as inhomogeneous distribution of prepolymer in the micro-cavities and inevitable post-treatment by degassing or centrifugation, which have restrained rapid fabrication of polymeric microneedles with consistent structural constitutions. Moreover, degassing or centrifugation of drug-loaded prepolymer may induce phase separation between the drugs and prepolymer, resulting in inhomogeneous encapsulation of bioactive molecules within the microneedle matrix.34,

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This problem may cause instable transdermal drug delivery, and

compromise the therapeutic efficacy or even induce severe drug resistance. Therefore, it is


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desirable to develop a novel strategy to realize homogeneous distribution of drug molecules in the polymeric matrix of microneedles rapidly and efficiently. As a natural fiber produced by silkworms, silk has been traditionally used as the source of threads and clothes for thousands of years.36 Silk fibroin has also been investigated recently as a promising material for biomedical applications attributed to its unique biocompatibility, controlled degradability and mechanical properties.36-38 Many nano- and microfabrication techniques with silk fibroin have been developed at various temperatures and atmospheric pressures.39,

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For

instance, silk fibroin was used as the matrix of biodegradable microneedles for controllable transdermal drug release.41-43 Silk fibroin was also used to construct three-dimensional (3-D) scaffolds for sustaining cell proliferation and differentiation. The pore size of these 3-D scaffolds is tunable from a few to hundreds of microns in diameter by precisely controlling the fibroin concentration, particularly appealing to tissue engineering applications.44-46 More importantly, the highly porous microstructure of fibroin scaffolds can direct liquid into internal cavities through capillary force spontaneously to achieve a higher level of saturation without degassing or centrifugation.47 This unique advantage of porous fibroin scaffolds may be utilized to achieve homogeneous distribution of drug-loaded prepolymer and thus improve the structural consistency of the microneedle matrix. Herein, we proposed a new PDMS-based micromold comprising a porous fibroin scaffold as a smart “sponge” for instantly absorbing drug-loaded liquid prepolymer during microneedle fabrication, which was uniformly distributed into the mold cavities without cumbersome degassing or centrifugation procedures. The fibroin-packed mold was obtained following a rapid freezedrying process.47 To customize the drug release kinetics, various amount of sucrose was introduced into PEGDA when preparing the prepolymer solution. PEGDA is a biocompatible and FDA-


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approved material for biomedical uses, and its monomer can be polymerized into a solid bulky structure initiated by UV irradiation. Previously, we demonstrated a sustained drug release capability of PEGDA-based microneedles triggered by polymer swelling under the physiological condition.48, 49 Meanwhile, sucrose is derived from abundant natural sources and widely used as a stabilizer or supplement in pharmaceutical industry. There are many benefits using sucrose, including biocompatibility, chemical simplicity, high solubility in water, and unique glass transition and rheological properties.50,

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In the present study, sucrose was used to adjust the

dissolvable components of microneedle matrix and the swelling rate of PEGDA in physiological interstitial fluid, thereby controlling the overall drug release kinetics (Figure 1). After casting the drug-loaded prepolymer solution onto the fibroin-packed PDMS micromold, PEGDA/sucrose microneedles were rapidly formed after UV light-induced polymerization. Two model drugs, including Rhodamine B (RhB) and doxorubicin (DOX), were encapsulated in various types of microneedles to investigate the drug release kinetics under a simulated physiological condition in vitro. The physicochemical and mechanical properties of the drug-loaded PEGDA/sucrose microneedles, including the morphology and mechanical strength, drug release rate and underlying mechanisms were systematically characterized, analyzed and discussed. Finally, we demonstrated the application of indocyanine green (ICG)-loaded microneedle patches on a mimic skin made of agarose hydrogel and the real skin of a live mouse. The homogeneity of the transdermally delivered therapeutics via microneedle patches was considerably enhanced and its photothermal effect was well maintained both in vitro and in vivo.


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Figure 1. Schematic illustrations of transdermal drug delivery through a microneedle system comprising PEGDA, sucrose and fibroin. The PEGDA backbone provides strong mechanical strength during skin penetration. Rapid drug release can be immediately triggered by dissolving of sucrose in the interstitial fluid of skin. The void space due to sucrose dissolution allows fluid permeation, which accelerates the swelling of PEGDA matrix and realizes subsequent phase of sustained drug release.

2. EXPERIMENTAL SECTION 2.1. Materials


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PDMS SYLGARD 184 silicone elastomer kit was purchased from Dow Corning Inc. Poly (ethylene glycol) diacrylate (PEGDA) (Mn=250), 2-hydroxy-2-methyl-propiophenone (HMP), sodium carbonate (Na2CO3), calcium chloride dehydrate (CaCl2·2H2O), Calcein AM sodium salt, indocyanine green (ICG), and agarose were obtained from Shanghai Aladdin Bio-Chem Technology Co., Ltd (China). Silkworm cocoons was provided by Institute of Sericulture and System Biology, Southwest University. Sucrose (>99.5%), rhodamine B (RhB, HPLC, ≥95%) and agarose (low gelling temperature) were supplied by Sigma-Aldrich (Germany). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Penicillin (10,000 unit·mL-1) /Streptomycin (10,000 ug·mL-1), Calcein AM and phosphate buffered saline (1×PBS) were obtained from Thermo Fisher Scientific (USA). Deionized (DI) water was collected from a MilliQ water purification system (Synthesis A10, Molsheim, France). Female BALB/c mice and KM mice were supplied by Chengdu Dossy Experimental Animals Co., Ltd. 2.2. Preparation of silk fibroin solution Aqueous silk fibroin solution from Bombyx mori cocoons was obtained according to a standard method as previously reported.52 Briefly, Bombyx mori cocoons was stripped from the silkworm and were subsequently sliced into thin pieces with 1-2 cm2 in size. The gum-like silk sericin protein was removed by incubating the cocoons in Na2CO3 solution (0.02 M) at 98~100oC for 40 min, followed by completely washing with DI water. After drying in a cabinet overnight, the silk fibers were dissolved in a mixed solution containing CaCl2, ethanol and water (molar ratio at 1:2:8) for 3 h at 70oC. A standard dialysis process was performed to eliminate small molecules and salts from the silk solution using Slide-a-Lyzer dialysis cassettes (Pierce, MWCO 8-12KD) for 2 days. Asprepared silk fibroin was further purified by centrifugation at 8000 rpm for 20 min, and the final silk fibroin solution was obtained at the concentration of 3–5% (w/v). All solutions were stored at


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4oC and ready for use. 2.3. Preparation of PDMS/fibroin mold The pristine PDMS mold was prepared by casting the PDMS prepolymer (elastomer: curing agent = 10: 1 by weight) onto a customized steel master (Micropoint Technologies PTE LTD, Singapore) with an array of 15 × 15 pyramid tips (400 μm in height and 250 μm in base length). After degassing in the vacuum for 1 h and thermal curation at 70oC for 2 h, the inversely replicated PDMS master mold was obtained by carefully peeling off from the metal substrate. Then, 200 μL as-prepared silk fibroin solution with various concentrations were casted onto the negative PDMS mold individually, followed by degassing in the vacuum to force the solution into the microcavities. After removal of excessive silk fibroin solution, the mold was frozen in a refrigerator at 40°C for 8 h. The fibroin were then lyophilized to yield a porous matrix as a scaffold in the microcavities of PDMS master mold. To observe the structure of fibroin scaffold, the porous matrix was removed from the PDMS mold and stained with Calcein AM (1.0 µM) for 10 min. After thoroughly washing with DI water, the fibroin scaffold was examined under a fluorescence microscope (Ex: 490 nm, Em: 515 nm). The porosity of the fibroin scaffold was measured following a liquid displacement method, and hexane was used as the displacement liquid.46 A pre-weighed sample was immersed in the hexane solution (volume of V1) for 10 min to allow the permeation of hexane into the porous scaffold structure. The total volume of the hexane and hexane-impregnated scaffold was denoted as V2. The volume difference (V2-V1) represented the volume of fibroin scaffold. The hexaneimpregnated scaffold was then removed from the system and the remaining hexane was recorded as V3. The volume of hexane within the fibroin scaffold was determined as V1-V3, representing the void volume of scaffold. The total volume of the scaffold can be expressed as V = (V2 - V1) + (V1 - V3) = V2-V3. Thus, the porosity of scaffold (ε) was calculated according to Equation (1):


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𝑉1 ― 𝑉3

ε = 𝑉2 ― 𝑉3 × 100% (1) 2.4. Fabrication of PEGDA/sucrose microneedle array The PEGDA/sucrose microneedle array was fabricated following a standard molding process of UV-induced polymerization. The PEGDA polymer solution was firstly mixed with HMP (0.5% w/w) as a photoinitiator. To fabricate the microneedle backing layer, a narrow cavity was created by placing a coverslip on top of the edges of two other cover slips (22 mm × 22 mm) supported by a glass slide to confine the geometry of the base. Then, native PEGDA prepolymer solution was carefully introduced into the cavity and the setup was exposed to a UV light source (wavelength = 365 nm, power intensity = 17 mW·cm−2) for 10 sec. After crosslinking, as-fabricated backing layer was carefully removed from the glass slide and dried in vacuum at 50 °C overnight. To fabricate the final microneedle patch, the modified prepolymer solution was prepared by adding various amount of sucrose into the PEGDA mixtures to obtain different weight ratios of sucrose:PEGDA at 1:20, 2:20 and 3:20. To load the model drugs, RhB or ICG was dissolved in the above prepolymer solution at a weight fraction of 0.3 wt% under gently mixing for 1 h to obtain a homogenous solution. Then, drug-loaded polymer was casted onto the PDMS/fibroin mold, where the cavities were filled spontaneously with prepolymer through capillary force. Pristine PDMS mold was concurrently tested for comparison. After excessive prepolymer solution was removed, previously developed PEGDA-based microneedle backing layer was placed onto the surface of PDMS/fibroin mold. Then, the mold was exposed to UV light (wavelength: 365 nm, power intensity: 17 mW·cm-2) for 40 sec. Finally, a drug-loaded patch with 15×15 microneedle array was peeled off from the mold and kept in a drying cabinet prior to use. 2.5. Characterizations of PEGDA/sucrose microneedle array


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Fluorescence of the encapsulated RhB (Ex: 553 nm, Em: 627 nm) was observed under a fluorescence microscope (Olympus, IX73, USA). Meanwhile, the morphology of microneedles was also examined using a scanning electron microscope (SEM, JSM-6510LV, Japan). Briefly, the microneedle array was sputtered with a thin layer of platinum (Pt). Then, the samples were placed into the vacuum chamber of SEM and observed from multiple angles using a three-axis nano-positioning system. The composition of PEGDA/sucrose microneedles were also characterized by analyzing their Fourier transform infrared (FTIR) spectrum using a spectrophotometer (Perkin Elmer, USA). 2.6. Mechanical property test The mechanical property of PEGDA/sucrose microneedles was characterized through a compression test using a universal testing machine (CMT4204, Suns Technology, China). The microneedle patch containing 15×15 needle arrays was fixed on a flat rigid aluminum plate under the vertically moving flat-head stainless cylindrical sensor prior to the test. The initial spacing between the microneedle tip to the transducer was set as 0.5 cm. Afterwards, the sensors moved vertically towards the aluminum stage at a constant rate of 0.5 mm per min. The sensor displacement and the corresponding resistance force on microneedles (i.e., stress-strain curve) was recorded from the moving sensors in contact with the microneedle tip. 2.7. Swelling properties of microneedles To investigate the swelling and dissolving properties of microneedles under aqueous condition, the microneedle patch with a 15×15 needle array were incubated in DI water (30 mL) for three days to enable thorough swelling and dissolving of microneedles. The weight of microneedle array before and after incubation were recorded as Wp and Ws, respectively. Subsequently, the sample


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was placed in a heating oven at 50oC for dehydration. After overnight, the weight of dry microneedle patch was measured as Wd. The morphology of microneedles after soaking was characterized using a scanning electron microscope (SEM, JSM-6510LV). The degree of swelling is calculated based on formula (2):

𝑄𝑠 =

𝑊𝑠 ― 𝑊𝑑 𝑊𝑑

× 100%

(2)

2.8. In vitro drug release from microneedles The drug loading capacity of PEGDA/sucrose microneedle array was determined based on the weight ratio of RhB in the prepolymer. Accordingly, the amount of drug loaded in each patch can be calculated from the patch weight and drug loading capacity. To evaluate the drug release behavior in vitro, PEGDA/sucrose microneedle patch was submerged in 30 mL of 1×PBS (pH=7.4) at 37oC. At predesignated time points, 2 mL of releasing medium was collected and stored at 4oC before analysis, followed by replenishing 2 mL fresh medium into the releasing system. The fluorescence calibration curves of RhB vs. concentration was determined by measuring the fluorescence spectrum of RhB (Ex: 553nm; Em: 627nm) using a microplate reader (SPARK 10M, Tecan). Thereafter, the released content of RhB was calculated accordingly based on the calibration curve using the aforementioned optical methods. On the other hand, low gelling temperature agarose hydrogel (2%, w/v) mimicking the physiological condition of a skin was created to further demonstrate the controllable drug release from PEGDA/sucrose microneedle array. Microneedles varying in the sucrose content were inserted into the hydrogel over a period of 120 h. At predesignated time points, the microneedle patches were removed from the hydrogel and the amount of drug delivered into the gel was determined based a dialysis method. Briefly, the hydrogel was loaded into a dialysis bag (MWCO=3500) and submerged in 30 mL of 1×PBS


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(pH=7.4) as the releasing medium at 60oC for 4 h. Then, the entire releasing medium was collected and stored at 4oC before analysis. Afterwards, fresh medium of the equivalent volume was replenished and similarly collected after another 4 h of incubation. The medium displacement was performed at least three times until the hydrogel became colorless and transparent. Thereafter, the released content of RhB was quantified accordingly based on above-mentioned fluorescence spectrophotometry. 2.9. Microneedle insertion test The insertion capacity of PEGDA/sucrose microneedles was firstly evaluated using an agarose hydrogel (1.4%, w/v) mimicking the physiological environment of skin.53 Then, the microneedles were inserted against the surface of hydrogel with a pressing force of 0.5 N per needle (in average) for 12 h. Thereafter, the microneedle patch was removed and the insertion site was imaged by a digital camera (Nikon D800E). The successful insertion rate was calculated as the number ratio of the pinhole with red color with the total microneedles on the patch. The fluorescence emission from the penetrated hydrogel was characterized under a fluorescence microscope (IX73, Olympus). Afterwards, the insertion capacity of PEGDA/sucrose microneedle arrays was tested on a sliced porcine skin. Specifically, the microneedles loaded with RhB were inserted into the porcine skin by pressing against their backing layer under a force of 0.5 N per needle (in average) using a universal testing machine. After removing the patch, the skin was washed with 1×PBS and wiped to ensure that there was no excessive RhB deposited on the skin surface. Then, the image of insertion site was captured with a digital camera (Nikon D800E), and the percentage of valid insertion sites was calculated based on the number of red spots and the total number of


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microneedles on each patch. The histological section was prepared to determine the penetration depth of each needle. Briefly, the penetrated skin area was excised using a scalpel. The skin sample was fixed in 10% formalin for 2 h and embedded in the tissue freezing medium at 20oC. Cryosection of 8 µm in thickness was prepared with a freezing microtome, stained with HematoxylinEosin (H&E) and examined using an optical microscope (IX73, Olympus, Japan). To demonstrate the effective delivery of a model drug for potential disease management, ICG-loaded PEGDA/sucrose microneedles were applied onto an agarose-based mimic skin, and the photoinduced therapeutic effect was characterized in vitro. 2.10. Biocompatibility The cytotoxicity study of drug-free PEGDA/sucrose microneedles were carried out using human umbilical vein endothelial cells (HUEVCs) via MTT cell viability assay. Briefly, HUVECs were seeded in a 96-well cell culture plate (1×104 cells per well) and incubated in DMEM culture medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37oC with 5% CO2 for 24 h. Then, PEGDA/sucrose microneedles were immersed in the cell culture medium for 24 h. Next, the previous medium was replaced by 200 µL of MTT solution (0.5 mg·mL-1) in DMEM. After further incubation for 4 h, the supernatant was aspirated carefully, and 200 µL DMSO was added to dissolve formazan crystals under gently shaking for 15 min. The optical absorbance of each plate well at 570 nm and 630 nm was measured using a microplate reader (SPARK 10M, TECAN). The cell viability was calculated according to the following formula (3):

Cell viability (%) =

(𝐴𝑏𝑠570 ― 𝐴𝑏𝑠630) 𝑜𝑓 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 (𝐴𝑏𝑠570 ― 𝐴𝑏𝑠630) 𝑜𝑓 𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠

(3)

For routine hematology analysis, PEGDA/sucrose (with fibroin) microneedles were applied onto the hair-removed skin of KM mice for 24 h. The whole blood samples collected from the


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ocular vessels of KM mice were subject to routine blood test to evaluate the biocompatibility and potential immunogenicity of PEGDA/sucrose microneedles using a hematology analyzer (BC5000VET, Mindray, China). The platelet (PLT), white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), and other blood indexes were measured postadministration at day 1, day 3 and day 7. 2.11 In vitro chemotherapeutic effect induced by the released DOX To investigate the potential of PEGDA/sucrose microneedles for chemotherapy, various PEGDAbased microneedle patches were fabricated by loading DOX into the needle matrix at a weight fraction of 0.5 wt%. In vitro cytotoxicity induced by the released DOX was evaluated using HeLa cells as the model cell line. Specifically, HeLa cells were seeded in 24-well plates (1 × 106 per well) and incubated for 24 h. Then, various microneedles were submerged into the culture medium and incubated with the cells for 24 h. Thereafter, the microneedles were removed from the wells, followed by incubating the cells for another 24 h, 48 h and 72 h. Cell viability were assessed by colorimetric MTT assays as described in the above section. 2.12. In vivo transdermal delivery on live mouse skin The animal experiments were carried out in compliance with the National Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) of Southwest University. Female BALB/c mice (5-7 weeks, 15 ± 1.2 g each) were utilized for investigating RhB delivery from PEGDA/sucrose microneedles, as well as skin recovery after administration in vivo. The drug-loaded microneedles were inserted into the skin of a live mouse by thumb pressing and retained for 10 min. To verify the successful penetration, the skin was sliced from the mice and stained with 0.4% trypan blue for 10 min and thoroughly washed with DI water. Images of the penetrated skin site with or without trypan blue staining were captured


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using a digital camera (Nikon D800E). To demonstrate the therapeutic potency of a real drug, various ICG-loaded microneedles were applied onto the bare (hair removed) skin of BALB/c mice for 2 min, followed by applying a NIR laser locally (808 nm, 2 W·cm-2). The body shell temperature was recorded using an infrared imaging system (Ti35, Fluke). 2.13. Statistical analysis The statistical analysis was performed based on one-way analysis of variance (ANOVA). A probability value (p-value) less than 0.05 (*p

Sucrose

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