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Tip-loaded fast-dissolving microneedle patches for photodynamic therapy of subcutaneous tumor
Accepted Manuscript Tip-loaded fast-dissolving microneedle patches for photodynamic therapy of subcutaneous tumor
Xiao Zhao, Xinfang Li, Peng Zhang, Jianwei Du, Youxiang Wang PII: DOI: Reference:
S0168-3659(18)30432-2 doi:10.1016/j.jconrel.2018.07.038 COREL 9398
To appear in:
Journal of Controlled Release
Received date: Revised date: Accepted date:
2 April 2018 10 July 2018 25 July 2018
Please cite this article as: Xiao Zhao, Xinfang Li, Peng Zhang, Jianwei Du, Youxiang Wang , Tip-loaded fast-dissolving microneedle patches for photodynamic therapy of subcutaneous tumor. Corel (2018), doi:10.1016/j.jconrel.2018.07.038
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Tip-loaded fast-dissolving microneedle patches for photodynamic therapy of subcutaneous tumor Xiao Zhao#, Xinfang Li#, Peng Zhang, Jianwei Du, Youxiang Wang
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MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310027, P.R.
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China.
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Abstract
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5-Aminolevulinic acid (ALA) based photodynamic therapy (PDT) is a modality for the treatment of cancers. However, due to its hydrophilicity and zwitterionic nature, the
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transdermal delivery of ALA is limited for the PDT of subcutaneous tumor. To address this problem, tip-loaded fast-dissolving microneedles made of sodium hyaluronate (HA)
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were fabricated by two casting method. 122 μg of ALA was loaded per microneedle patch and mainly distributed in the tips, which could improve the utilization of drug and avoid the waste of drug residue in the base of microneedle patch after use. The HA
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microneedles could pierce stratum corneum with insertion depth about 200 μm in
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isolated skin. After insertion, HA microneedles were rapidly dissolved to release the encapsulated drug to improve patients’ convenience and compliance. Importantly, in a subcutaneous mouse tumor model established in BALB/c nude mice, the PDT efficacy of ALA-loaded HA microneedle group was much better than ALA injection group in spite of a relatively lower ALA dose with HA microneedles. The tumor inhibition rate of
#
X. Zhao and X. Li contributed equally to this work.
Corresponding author. E-mail address: [email protected](Y.X. Wang)
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ALA-loaded HA microneedle group (containing 0.61 mg of ALA) was up to 97%, while the tumor inhibition rate of ALA injection group (containing 1.65 mg of ALA) was just 66%. In addition, microchannels created by microneedle patch were quickly recovered
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within 3 h after insertion. Overall, the tip-loaded fast-dissolving HA microneedle patch
subcutaneous tumor in an efficient and safe manner.
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with ALA as drug was promising and might improve topical PDT efficacy of
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Key words: Photodynamic therapy; Dissolving microneedles; Subcutaneous tumor; HA
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1. Introduction
Photodynamic therapy (PDT) is a favorable method for the treatment of various
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tumors and non-neoplastic diseases[1, 2]. In PDT, a combination of the photosensitizer, oxygen and light at a fixed wavelength generates highly cytotoxic reactive oxygen species (ROS) to kill selected cells, obtaining efficient therapeutic outcome with
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minimal side effect[3]. 5-Aminolevulinic acid (ALA), a precursor of second generation
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photosensitizer, can be converted to endogenous protoporphyrin IX (PPIX) after a series of reactions in mitochondria[4]. ALA-based PDT has been widely applied for a number of skin lesions, such as actinic keratoses[5], basal cell carcinoma[6, 7] and squamous cell carcinomas[8]. Compared with systemic administration, topical administration is more favorable, since photosensitization only occurs locally at the site of treatment and avoids severe skin photosensitivity[8-10]. However, the transdermal penetration of ALA is
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limited by its physiochemical characteristics, such as hydrophilicity and zwitterionic nature[11]. To increase the transdermal delivery of ALA, plenty of chemical and physical techniques are explored, such as penetration enhancer[11], lipophilic derivatives[12],
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nanoemulsions[13], iontophoresis[14], sonophoresis[15] and microneedles[16]. In contrast to
17]
. Microneedles are usually considered as a
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performance and little side effect[9,
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other methods, microneedle technology gets a widespread attention for excellent
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synergistic combination of transdermal patch and hypodermic needle, which can pierce the protective barrier of the skin, stratum corneum, to promote the transdermal drug 19]
. Through the reversible microchannels created in the skin, the
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delivery[18,
penetrability of skin-impermeant drugs is enhanced. Furthermore, microneedle insertion
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causes minimal pain compared with injection needles, because the limited insertion depth of microneedles will not stimulate nerves in the skin[20].
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Microneedle-mediated ALA-based PDT has been studied to explore its applications for treating tumors. Donnelly et al. investigated the potential of solid
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silicon microneedles for treatment of tumor via poke-and-patch method[9]. This strategy accelerated the penetration of ALA through the murine skin and induced higher level of PPIX in vivo experiments. Recent work by Gill et al. showed ALA-coated stainless microneedles promoted the transdermal delivery of ALA and its inhibition effect on subcutaneous tumor was around 57% with a dose of just 1.75 mg of ALA, while the topical cream including 5 mg of ALA was not capable of suppressing tumor growth [17].
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Nevertheless, no matter poke-and-patch method or coated solid microneedles, these inorganic or metal microneedles suffer from some issues. The microchannels created by microneedles in poke-and-patch method reseal quickly after removing microneedle patches, which precludes the further penetration of the encapsulated drug[18]. The drug 21]
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loading of microneedles by coating or dipping is low[20,
. In addition, inorganic
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microneedles are brittle to rupture in the skin, which may cause safety issues[22]. Mental
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microneedles must be removed and threw away as sharp biohazardous waste after use[23].
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Therefore, a kind of microneedle patch with sufficient mechanical strength, safety, easy
microneedle patch is one of them.
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fabrication and relatively larger drug loading capacity is needed, and the polymer
In recent years, polymer microneedles have been widely used to transdermally
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deliver a variety of drugs, especially hydrophilic drugs and macromolecular drugs[24, 25]. To date, many polymer materials have been utilized to fabricate polymer microneedles, such as sodium hyaluronate (HA)[26,
27]
, chitosan[22,
28]
, polyvinyl alcohol[29],
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polyvinylpyrrolidone[30, 31] and polylactic acid[32]. Polymer microneedles have sufficient
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mechanical strength to puncture the stratum corneum and maintain the stability of drugs. During application, although some polymer microneedles may rupture in the skin, it is safe because of good biocompatibility and biodegradability of polymer materials[21, 30]. HA is a sort of biocompatible polysaccharide and an important ingredient of the skin, which has been approved by the U.S. Food and Drug Administration (FDA) to fill soft tissue damage in 2003. HA can promote cell movement and proliferation, inhibit cell
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differentiation and regulate the conglutination of cell and extracellular matrix. And the degradation product, monosaccharide, can also take part in metabolism circulation. In this study, we utilized HA to fabricate tip-loaded fast-dissolving polymer microneedle
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patch to enhance the transdermal delivery of ALA for PDT of subcutaneous tumor. The basic properties of HA microneedles, such as mechanical strength, insertion depth,
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dissolution behavior and drug release were investigated systematically. Next, in vivo
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PDT efficacy of ALA-loaded HA microneedle patch was examined in a subcutaneous
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mouse tumor model using human oral epidermoid carcinoma (KB) cells. Finally, skin recovery was simply tested in BALB/c nude mice. The tip-loaded fast-dissolving HA
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microneedle patch could adjust drug loading efficiency easily and might improve
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2. Materials and methods
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topical PDT efficacy of subcutaneous tumor in an efficient and safe manner.
2.1 Chemicals
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Sodium hyaluronate (HA, Mw approximately 10kDa) was supplied by Zhenjiang Dong Yuan Biotech Co., Ltd. (Zhenjiang, China). Rhodamine 6G, 5-aminolevulinic acid
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(ALA), sodium sulfite, and 2,4,6-picrylsulfonic acid solution (TNBS, 5% w/v) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Sodium dihydrogen phosphate dihydrate was purchased Huzhou Hushi Chemical Reagent Co., Ltd. (Huzhou, China). Polydimethylsiloxane (PDMS, Sylgard 184) and the optimum cutting temperature (OCT) compound were purchased from Dow Corning (Wiesbaden, Germany) and Sakura Finetek Japan Co., Ltd. (Hamacho, Japan), respectively. PDMS
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molds of microneedles (with about 1000 μm height, 300 μm width at base, 1500 μm needle pitch and 5×5 array) were purchased from Micropoint Technologies Pte. Ltd. (Singapore). Ultra-pure deionized water (Millipore, Direct Q® 3 UV) was used for all
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the experiments. All other chemicals were of analytical grade. 2.2 Cell culture
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Human oral epidermoid carcinoma (KB) cells were provided by China Centre for
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Typical Culture Collection and cultured in RPMI 1640 medium containing 10%
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heat-inactivated fetal bovine serum, 1% penicillin and 1% streptomycin under 5% CO2 at 37 ℃. All experiments were conducted in the logarithmic phase of cell growth.
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2.3 Animals
All animal experiments were carried out according to the “Principles of Laboratory
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Animal Care” (NIH publication no. 86-23, revised 1985) and the guidelines for Animal Care and Use Committee, Zhejiang University. Healthy male Sprague-Dawley rats (SD
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rats, 6 weeks old, weight around 200 g) and male BALB/c nude mice (5 weeks old, weight around 15 g) were supplied by the animal center of Zhejiang Academy of
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Medical Sciences and Shanghai SLAC Laboratory Animal Co., Ltd., respectively. The abdominal skin of Sprague-Dawley rat (SD rat) with hair shaved was pre-prepared and kept in the -80 ℃ super cold refrigerator for in vitro skin insertion tests. To set up the tumor model, KB cells in PBS buffer (pH7.4) were injected (100 μL, 2.2 × 106 cells/mouse) subcutaneously into the right flank of BALB/c nude mice. 2.4 Fabrication of tip-loaded fast-dissolving HA microneedle patch
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The tip-loaded fast-dissolving HA microneedles were fabricated via two-casting method (Fig. 1)[20,
33]
. Briefly, PDMS inserts in 50 mL corning tubes were first
manufactured. Next, drug-loaded casting solution containing 1 g of HA, 92.8 mg of
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ALA and 2 mL of deionized water were prepared. Following that, 50 μL of the prepared drug-loaded solution was poured into each PDMS mold and then placed in 50 mL
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corning tubes containing PDMS inserts. After that, they were centrifuged (Heraeus
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Biofuge Stratos Centrifuge, Thermo scientific, USA) in a swinging bucket rotor at 4,500
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rpm for 25 min to fill the cavities of the PDMS mold and form the tips of microneedles. Superfluous solution left on the surface of PDMS molds was removed by pipette. The
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molds were re-centrifuged for 3 min to promote compaction. A pure HA gel solution (50 wt.%) was added to the surface of the molds and centrifuged at 4,500 rpm for 45min to
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form the base of microneedles. Then, these molds were dried in an oven at 37 ℃ overnight. Finally, the ALA-loaded HA microneedle patches were gently detached from
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the molds and kept in the desiccator containing silica gel. The rhodamine 6G-loaded HA microneedle patches were fabricated by the same production process. The microneedle
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patches were stored in the dry and dark environment for further use. 2.5 Morphology and drug loading of microneedles The morphology of ALA-loaded HA microneedles was examined by a stereoscope (VHX-2000, Keyence, Japan). The sizes of microneedles were analyzed by Image J. Details of dimensions were illustrated, including the height, tip diameter, width of base and needle pitch of ALA-loaded HA microneedles. To observe the distribution of drug
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in microneedle patches, rhodamine 6G, a red fluorescent dye, was used as a model drug. The ALA-loading content in the HA microneedle patch was determined using TNBS method according to a previously reported literature[34]. In brief, each
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ALA-loaded HA microneedle patch was fully dissolved in 1mL of deionized water to release all the encapsulated ALA, then the solution was centrifuged at 12000 rpm for 5
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min to collect the supernatant. 600 μL of mixed solution with equal volume of obtained
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supernatant, borate buffer (0.1 mol/L, pH8.4), and TNBS solution (0.1% w/v) was
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reacted at 50 ℃ for 40 min, then 200 μL of sodium dihydrogen phosphate solution (0.1mol/L) containing 2×10-6 mol of sodium sulfite was added to the mixed solution to
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stop the reaction. After that, the mixed solution was kept at room temperature for 20 min, and ultraviolet-visible spectrophotometry (UV-2550, Shimadzu, Japan) was
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utilized to analyze the mass of released ALA according to the standard curve. 2.6 Mechanical property of microneedles
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A universal testing machine (5543A, Instron, USA) was used to preliminarily assess the mechanical property of ALA-loaded HA microneedles as previously
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reported[28]. Each ALA-loaded HA microneedles was put on a stainless steel plate with tips upward. The force, perpendicular to the plate, was applied by a moving sensor to press ALA-loaded microneedles at the constant speed of 200 μm/s. The machine recorded the force of moving sensor as a function of ALA-loaded HA microneedles displacement. In addition, initial and final morphology of ALA-loaded HA microneedles during compression test were obtained using a stereoscope.
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2.7 In vitro skin insertion tests 2.7.1 In vitro skin insertion ability of microneedles To further estimate the strength of microneedles, the isolated abdominal skins of
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SD rats were first taken out from refrigerator and soaked in 0.9 wt.% sodium chloride solution to reach the room temperature, then the water on the skin surface was sucked
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by filter papers. Next, these skins were fixed on a foam board before the experiment.
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Following that, the rhodamine 6G-loaded HA microneedle patch was inserted into the
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skins using a thumb for 2min. After insertion, the rhodamine 6G-loaded HA microneedle patch was removed. The skin surface was rubbed carefully by presoaked
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cotton swab to make sure that no rhodamine 6G was remained on the skin surface. Then these skins were examined by a digital camera to calculate the insertion ratio, which
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was calculated by dividing the number of remained red dots on the skin surface by the total number of needles of microneedles. Finally, the skin was excised to determine the
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insertion depth by frozen tissue sections. And the insertion depth was defined as the average depth of holes created by the microneedle patch.
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The samples of frozen tissue sections were prepared as follows. Microneedle insertion area was separated from the skin with a scalpel. The isolated skin sections were put into -80 ℃super cold refrigerator for about 2 min, then the sample was embedded in OCT compound in a cryostat mold. The frozen samples were cut into 10 μm thick sections using a cryotome (CryoStar NX50, Thermo scientific, USA) and then placed on adhesion microscope slides (Citotest Labware Manufacturing Co., Ltd).
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Finally, the skin sections were photographed under a microscope (BX61, Olympus, Japan). Meanwhile, to characterize the permeation of drug in the vertical direction of
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full-thickness skin, the rhodamine6G-loaded HA microneedle patch was inserted into the abdominal skins of SD rats for 2 min as described above. After that, the skin was put
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on a microscope slide and analyzed with a confocal laser scanning microscope (TS SP5,
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Leica, Germany) at the excitation wavelength of 526 nm. Pictures were collected in the
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xy-plane. Scanning was operated once at the interval of 10 μm from skin surface through the vertical direction of xy-plane, and the starting position of scanning of the
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skin surface (z= 0 μm) was viewed as the plane with the brightest fluorescence. 2.7.2 In vitro dissolution of microneedles
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The dissolution rate of the ALA-loaded HA microneedle patch was investigated on the isolated abdominal skins of SD rats. The SD skins were pretreated as described
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above, then ALA-loaded HA microneedle patches were applied on the skins using a thumb for different time. At the indicated time points, ALA-loaded HA microneedle
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patches were removed from the skin and observed by a stereoscope. And the size variations of microneedles were analyzed by Image J. 2.7.3 In vitro transdermal delivery of drug In vitro release of ALA from ALA-loaded HA microneedle patches was investigated by Franz diffusion cell (RYJ-12B, Huang Hai Yao Jian, China) as previous report[35, 36]. The isolated SD skins were first pretreated as described above. ALA-loaded
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HA microneedle patch was then inserted into the skin using a thumb for 2 min and fixed to the skin by medical adhesive tape. Next, the skin with microneedle patch was mounted onto the Franz cell with dermal side facing the receptor chamber. The permeation area of Franz cell was about 2.39 cm2. The receptor medium (7.1 mL of
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PBS, pH7.4) was maintained 32 ℃in an incubator and continuously stirred 300 rpm. At
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predetermined intervals, samples (0.6 ml) were withdrawn from the receptor chamber
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and replaced with an equal volume of fresh PBS buffer. Subsequently, the samples
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extracted from cells were stored at -20 ℃ until analysis. The cumulative permeation amount of ALA from microneedles was determined using TNBS method as discussed
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above. 2.8 In vivo experiments
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2.8.1 Antitumor efficacy of microneedles To evaluate the photodynamic therapeutic effect of ALA-loaded microneedle patch
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in vivo, treatment was begun when the tumor volume reached around 130 mm3. Tumor bearing mice were randomly divided into four groups (n=4 per group): (1) Untreated
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group, in which the mice were untreated; (2) Only light group, in which the mice were just treated with the light exposure (635 nm, 450 mW) for 10 min; (3) ALA injection + light group, in which the mice were first injected 100 μL of heat-inactivated PBS buffer (pH 7.4) containing 1.65 mg of ALA through caudal vein, and then under light exposure (635 nm, 450 mW) for 10 min after 4 h post injection; (4) ALA microneedles + light group, in which the mice were first treated with ALA-loaded microneedle patches (122
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μg of ALA per microneedle patch, 5 patches), and then under light exposure (635 nm, 450mW) for 10 min after 4 h post insertion. Each microneedle patch was inserted into skin for 10 min and then removed. The therapeutic effect was examined by the change of tumor volume. Tumor sizes were measured with an electronic digital caliper, and
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tumor volume was calculated using the formula: tumor volume = 0.5 × length ×
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width2[37]. After 14 d therapy, tumor bearing mice were sacrificed to obtain the images
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and weights of tumors of each group to accurately estimate the therapeutic efficacy, and
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the tumor inhibition rate was calculated by dividing the excised tumor volume of the treatment group by the excised tumor volume of the control group.
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2.8.2 Skin recovery after microneedle insertion
Skin recovery ability after microneedle insertion was simply evaluated on the skin
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of BALB/c nude mice in vivo. Briefly, the ALA-loaded HA microneedle patch was inserted into the back skin of BALB/c nude mice for 2 min, then the microneedle patch
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was removed from the skin surface. Images of the insertion area after microneedle treatment were obtained by a digital camera at 0 min, 10 min, 30 min, 1 h, 3 h, and 17 h
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after insertion.
2.9 Statistical analysis For this study, all values were presented as their mean ± standard deviation. Statistical differences were considered to be significant when p < 0.05. 3. Results and discussion 3.1 Morphology and drug loading of microneedles
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To prepare the tip-loaded fast-dissolving HA microneedles, a two-casting fabrication process was adopted. As shown in the Fig. 1, the mixed HA solution containing ALA was first poured into PDMS molds to form the tips of microneedles,
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then the blank HA gel solution without ALA was cast to PDMS molds to form the base of microneedles.
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As shown in the Fig. 2, the fabricated microneedles had the identical structure of
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pyramidal shape. ALA-loaded fast-dissolving HA microneedles consisted of 25 (5×5)
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pyramidal needles in Fig. 2(a). The height, tip diameter, width of base and needle pitch were 907 + 20 μm, 12 + 5 μm, 309 + 16 μm and 1123 + 2 μm, respectively. It was
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approximately 9.3% reduction in height compared to PDMS mold, which might be caused by water evaporation during drying process[38]. And the base area was a thick
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and rigid board. Thus the force acting on the baseplate could be uniform to facilitate the microneedles insert into the skin by hand both in vitro and vivo. Meanwhile, rhodamine
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6G was chosen as model drug to visualize the distribution of drug in the microneedles. As shown in Fig. 2(b), although the color of the base of rhodamine 6G-loaded HA
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microneedles was slightly deepened, rhodamine 6G was mainly dispersed in the tips of microneedles. It indicated this design, tip-loaded microneedle patch, could improve the utilization of drug and avoid the waste of drug residues in the base of microneedle patch after treatment, and highlight the unique advantage of two-casting method to prepare microneedles. It was worth of noting that the color of the base might be attributed to the diffusion of rhodamine 6G during preparation. And ALA content in ALA-loaded HA
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microneedles was determined by TNBS method. The loading amount of ALA was 122+8 μg per microneedle patch. 3.2 Mechanical property of microneedles
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It’s necessary for polymer microneedles to have sufficient mechanical strength to pierce the stratum corneum. Factors, such as tip diameter, geometry, material
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composition and aspect ratio, may affect the strength of polymer microneedles[38, 39].
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Pyramidal microneedles were more robust than conical microneedles due to the unique
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property to the larger cross-sectional area of pyramidal microneedles at the same base width or diameter[28]. Therefore, pyramidal microneedles were chosen in this work.
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Results from mechanical compression test (Fig. 3a) showed that the force acting on ALA-loaded HA microneedles elevated with the increase of the displacement of
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microneedles without a distinct transition point. ALA-loaded HA microneedles (Fig. 3b) undertook progressive deformation without notable separation during compression test. The force reached 0.6 N/needle when the displacement was 0.2 mm, which was
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41]
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obviously comparable or even superior to performances from previous reports[28, 38, 40,
3.3 In vitro skin insertion tests 3.3.1 In vitro skin insertion ability of microneedles To further examine the strength of microneedles, in vitro insertion test was carried out to obtain the skin insertion ratio and insertion depth using the isolated abdominal skin of SD rats with hair shaved. The rhodamine 6G-loaded HA microneedle patch was
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applied to the skin for 2 min and subsequently removed from the skin. The red dots (Fig. 4a) that couldn’t be wiped out on the skin surface was rhodamine 6G left in the skin, indicating that microneedles had successfully inserted into the skin. Furthermore, the
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histological results (Fig. 4b) further verified the result, and it showed red dye had spread from the holes created by microneedles to the surrounding tissue.
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As shown in the Fig. 4(a) and Fig. 4(b), the skin insertion ratio was 100%, and the
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insertion depth was 218 + 52 μm, which was far shorter than the height of microneedles
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(approximately 907 μm). The proper reason could be ascribed to two aspects: Firstly, it might be the result of elasticity of the skin[38]. Secondly, when microneedles were
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inserted into the skin, HA could adsorb physiological fluid and the tips dissolved quickly. The insertion process accompanied with dissolution. Thus the insertion depth
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was much shorter than the height of microneedles. The microneedles could pierce through the stratum corneum (the thickness was10-20 μm) and reach the dermis layer
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without piercing through the skin.
Meanwhile, in order to determine the penetration depth of encapsulated drugs in
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the skin, the skin treated by rhodamine 6G-loaded HA microneedle patch was observed using a confocal microscope. The skin insertion site was scanned at varying depth in the direction perpendicular to the skin surface. As shown in the Fig. 4(c), after insertion for 2 min, the penetration depth of rhodamine 6G could reach 600 μm. The histological section in Fig. 4(b) suggested the insertion depth was only 218±52μm. So in the Fig. 4(c), we could see red fluorescence from the surface to the depth of 200μm, which was
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the position of tips actually. On the other hand, the microneedles had the identical structure of pyramidal shape in this research. The drug content near the base area of the microneedles was much larger than that in tips. It was normal to observe that the red
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fluorescence intensity at the depth of 200μm was less than that near surface. Furthermore, red fluorescence could rapidly spread from application site to surrounding
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tissue. All results indicated the microneedles had efficient strength to perforate the
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stratum corneum and delivered the encapsulated drugs directly into dermal tissue.
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3.3.2 In vitro dissolution of microneedles
HA was commonly used as the matrix material of dissolving microneedles owing
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to its high hydrophilicity and biocompatibility[20,
41]
. The dissolution behavior of
ALA-loaded HA microneedle patch was investigated. As shown in Fig. 5, the
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dissolution rate was nonlinear. In the first 1 min, the height of microneedles was reduced by approximately 42%, indicating a fast initial dissolution. After skin insertion
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for 4min, the height of microneedles was reduced by approximately 75%. Subsequently, the dissolution rate of microneedles became relatively slow. Approximately 80% of their
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initial height was reduced after insertion for 6 min. According to Fig. 2(b), this part contained the majority of drug. It was worth noting that the dissolution height was higher than insertion depth. As we mentioned ahead, HA could adsorb physiological fluid and the tips dissolved quickly. The insertion process accompanied with dissolution. So the actual dissolution part of microneedles was obviously longer than the penetration depth. These results demonstrated that ALA-loaded fast-dissolving microneedles were
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successfully constructed, and it could reduce the treatment time and improve patients’ convenience and compliance. In the following tumor growth inhibition experiment, we chose insertion time for 10 min to fully release the encapsulated drug.
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3.3.3 In vitro transdermal delivery of drug Drug release behavior of microneedles in vitro was examined by Franz diffusion
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cell. ALA-loaded HA microneedles were inserted into the isolated abdominal skin of SD
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rats, and drug release was monitored for 4 h. As shown in Fig. 6, in the first 5 min, the
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release of ALA from HA microneedles exhibited a burst release. HA microneedles were rapidly dissolved to release the encapsulated ALA, and the percentage of cumulative
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release was about 40% within the first 5 min. After that, the release of ALA exhibited a slow release over time. After 60 min, almost all of the encapsulated ALA was released,
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which was consistent with the published literature[41]. In vitro skin insertion tests demonstrated that the microneedles had efficient
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strength to pierce the stratum corneum，dissolved quickly, released the encapsulated drug to promote the transdermal drug delivery.
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3.4 In vivo experiments
3.4.1 Antitumor efficacy of microneedles To evaluate the therapeutic efficacy of ALA-loaded HA microneedles, tumor model was established in BALB/c nude mice using KB cells. When the tumor volume of mice grew to approximately 130 mm3, the mice were divided into four groups, and labeled as the “Untreated” group, the “Only light” group, the “ALA injection + light” group and
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the “ALA microneedles + light” group, respectively. The “Untreated” group was served as the control group. The therapeutic efficacy of PDT using subcutaneous tumor was assessed by the
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tumor volume change[42]. As shown in Fig. 7(a), the tumor volume of the control group increased rapidly, and the tumor volume was about 455% of the initial volume at day 7.
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Compared with the control group, the “Only light” group could slow tumor growth to
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certain extent. The tumor volume was about 355% of the initial volume at day 7, which
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indicated the effect of light was relatively limited. The therapeutic efficacy of the “ALA microneedles + light” group was the best, and tumor size of mice presented an obvious
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decrease trend. The tumor was about 44% of initial tumor volume in the “ALA microneedles + light” group after treatment for 7 d. Given that the tumor might recur,
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the tumor-bearing mice were examined for another week without any further treatment. After 2 weeks’ treatment, in the “ALA microneedles + light” group, the tumor of only
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one mouse relapsed in the second week while the tumor of three others remained invisible. Representative images of tumor-bearing mice of different groups at day 14
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were shown in Fig. 7(b).
After 2 weeks’ treatment, the mice were sacrificed to obtain the images and tumor weights s of each group to accurately estimate the therapeutic efficacy. As shown in Fig. 7(c) and Fig. 7(d), tumor grew most rapidly in the control group, and the average weight of excised tumor was about 1.65 g. In the “Only light” group, the final inhibition efficacy was about 50%, it might be attributed to high light intensity (635nm, 450mW,
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10 min). In the “ALA injection + light” group (containing 1.65 mg of ALA), its final inhibition efficacy was about 66%, slightly higher than only light group. However, the final inhibition efficacy of the “ALA microneedles + light” group (containing 0.61 mg
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of ALA) was the highest up to 97%. The PDT efficacy of ALA-loaded HA microneedles group was better than ALA
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injection group in spite of a lower ALA dose (microneedles: 0.61 mg vs injection:1.65
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mg ALA). It might be the following reasons. In the case of injection group, ALA
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injected through caudal vein would circulate around the body before reaching the tumor tissue, diminishing the conversion rate of ALA into PPIX. Therefore, it would be
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difficult to exploit the drug to its fullest. Instead, ALA-loaded HA microneedle patches were directly applied to the skin over the subcutaneous tumor in situ, which would have
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higher utilization of drugs and improve the therapeutic efficacy. 3.4.2 Skin recovery after microneedle insertion
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Lastly, the skin recovery rate was simply evaluated by observing the surface appearance of skin damage. The ALA-loaded microneedle patch was applied to the back
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skin of BALB/c nude mice in vivo and subsequently removed 2 min later. After treatment, there were no obvious side effect except for some visible micro-pores and local erythema in insertion site (Fig. 8). After 3 h post insertion, the majority of erythema rapidly faded away, and microchannels created by microneedles were almost resealed. Resealed microchannels were capable of reducing the risk of infection and entry of pathogens[18,
21]
. What’s more, the insertion skin reverted to its original
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condition after 17 h post insertion, indicating that the skin had good recovering ability after microneedle treatment. In vivo PDT and skin recovery experiment demonstrated the tip-loaded
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fast-dissolving HA microneedle patch was an efficient and safe way for transdermal drug delivery. However, considering the differences between mice skin and human skin,
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further experiments still need to be conducted systematically to prepare for clinical
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studies in the future.
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Conclusion
This study demonstrated that the tip-loaded fast-dissolving HA microneedle patch
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with ALA was an efficient and safe way for PDT of subcutaneous tumor. Tip-loaded HA microneedles were fabricated by two-casting method. Each microneedle patch contained
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122 μg of ALA, which were mainly distributed in the tips of needles. The insertion ratio and insertion depth were 100% and approximately 200 μm, respectively. After skin
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insertion for 4 min, the dissolved height percentage of microneedles was about 75%. These results suggested the microneedles had efficient strength to pierce the stratum
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corneum，dissolved quickly, released the encapsulated drug to promote the transdermal drug delivery. In the subcutaneous mouse tumor model, the PDT efficacy of ALA-loaded HA microneedles group was better than ALA injection group. The tumor inhibition rate of ALA-loaded HA microneedles group (containing 0.61 mg of ALA) was up to 97%, while the tumor inhibition rate of ALA injection group (containing 1.65 mg of ALA) was just 66%. In addition, microneedle insertion caused no obvious side
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effect, and the skin had good recovering ability after microneedle treatment. Therefore, our study suggested that the tip-loaded fast-dissolving HA microneedle patch with ALA was promising and might improve topical PDT efficacy of subcutaneous tumor in an
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continuous production capacity still remained a challenge.
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efficient and safe manner. Considering the potential application in clinical, the
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Acknowledgements
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This work was financially supported by the Natural Science Foundation of Zhejiang Province (No. LY18E03001)， Zhejiang science and technology program
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(2016C04002) and the National Natural Science Foundation of China (No. 21474087).
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of Controlled Release, 161 (2012) 933-941. [42] M.C. Chen, Z.W. Lin, M.H. Ling, Near-Infrared Light-Activatable Microneedle System for Treating Superficial Tumors by Combination of Chemotherapy and
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Figure Captions Fig.1 Schematic illustration of the process to fabricate the tip-loaded fast-dissolving HA
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microneedle patch.
Fig.2 Stereomicroscopy images of ALA-loaded fast-dissolving HA microneedle patch
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(a) and rhodamine 6G-loaded fast-dissolving HA microneedle patch (b). Diagrammatic
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representation of the microneedle patch and its geometric parameters (c).
Fig. 3 Mechanical behavior of ALA-loaded HA microneedle patch. (a) Force was
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measured as a function of microneedles displacement, and the inset illustrates the schematic diagram of the measuring device. (b) Initial and final images of ALA-loaded
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HA microneedles were obtained during compression test. Each data point represents
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mean ± standard deviation (n = 3).
Fig. 4 Skin insertion ability of rhodamine 6G-loaded HA microneedle patch.
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Bright-field micrograph (a), histological section (b) and CLSM images (c) of the mouse skin were photographed after microneedle insertion for 2 min(white scale bar: 500 μm).
Fig. 5 Dissolution of ALA-loaded HA microneedle patches after their application to the skin in vitro. Dissolution data (a) and images (b) of ALA-loaded HA microneedle patches after insertion. Each data point represents mean ± standard deviation (n = 6).
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Fig. 6In vitro drug release behavior of ALA-loaded HA microneedle patches. Schematic diagram of Franz diffusion cell used (a). In vitro drug release profiles (b). The partial
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enlarged drawing of the dotted box (c). The loading amount of ALA was about 122 μg
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per patch. Each data point represents mean ± standard deviation (n = 4).
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Fig. 7 Photodynamic therapy of subcutaneous tumor. (a) Tumor growth curves of
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different groups of tumor-bearing mice after treatment. (b) Representative images of different groups of tumor-bearing mice after 14 days’ treatment. (c) Weight of excised
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tumor of each group after 14 days’ treatment. (d) Images of excised tumor of each group after 14 days’ treatment. Treatment was started when the tumor volume reached the size
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of about 130 mm3. Tumor bearing mice were randomly divided into four groups: (Ⅰ) Untreated group, in which the mice were untreated; (Ⅱ) Only light group, in which the mice were just treated with the light exposure (635 nm, 450 mW) for 10 min; (Ⅲ) ALA
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injection + light group, in which the mice were first injected 100 μ L of
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heat-inactivated PBS containing 1.65 mg of ALA through caudal vein, and then under red light exposure (635 nm, 450 mW) for 10 min after 4 h post injection; (Ⅳ) ALA microneedles + light group, in which the mice were first treated with ALA-loaded microneedle patches (122 μg of ALA per microneedle patch, 5 patches), and then under light exposure (635 nm, 450mW) for 10 min after 4 h post insertion. Each
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microneedle patch was inserted into skin for 10 min and then removed. Each data point represents mean ±standard deviation (n = 4).
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Fig. 8 The skin recovery after microneedle treatment. The representative images of mice
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min, 10 min, 30 min, 1 h, 3 h and 17 h post treatment.
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treated with a microneedle patch for 2min. Skin puncture marks were photographed at 0
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Fig. 1. Schematic illustration of the process to fabricate the tip-loaded fast-dissolving
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HA microneedle patch.
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Fig. 2. Stereomicroscopy images of ALA-loaded fast-dissolving HA microneedle patch (a) and rhodamine 6G-loaded fast-dissolvingHA microneedle patch (b). Diagrammatic representation of the microneedle patch and its geometric parameters (c).
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Fig. 3. Mechanical behavior of ALA-loaded HA microneedle patch. (a) Force was measured as a function of microneedles displacement, and the inset illustrates the
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schematic diagram of the measuring device. (b) Initial and final images of ALA-loaded HA microneedles were obtained during compression test. Each data point represents
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mean ± standard deviation (n = 3).
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Fig. 4. Skin insertion ability of rhodamine 6G-loaded HA microneedle patch. (a)Bright-field micrograph, (b) histological section and (c) CLSM images (scale bar:
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500 μm) of the mouse skin were photographed after microneedle insertion for 2 min.
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Fig. 5. Dissolution of ALA-loaded HA microneedle patches after their application to the
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skin in vitro. Dissolution data (a) and images (b) of ALA-loaded HA microneedle
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patches after insertion. Each data point represents mean ± standard deviation (n = 6).
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Fig. 6.In vitro drug release behavior of ALA-loaded HA microneedle patches. Schematic diagram of Franz diffusion cell used (a). In vitro drug release profiles (b).
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The partial enlarged drawing of the dotted box (c). The loading amount of ALA was
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about 122 μg per patch.Each data point represents mean ± standard deviation (n = 4).
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Fig. 7. Photodynamic therapy of subcutaneous tumor. (a) Tumor growth curves of different groups of tumor-bearing mice after treatment. (b) Representative images of
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different groups oftumor-bearing mice after 14 days’ treatment. (c) Weight of excised
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tumor of each group after 14 days’ treatment. (d) Images of excised tumor of each group after 14 days’ treatment. Treatment was started when the tumor volume reached the size of about 130 mm3. Tumor bearing mice were randomly divided into four groups: (Ⅰ) Untreated group, in which the mice were untreated; (Ⅱ) Only light group, in which the mice were just treated with the light exposure (635 nm, 450 mW) for 10 min; (Ⅲ) ALA injection + light group, in which the mice were first injected 100 μL of heat-inactivated PBS containing 1.65 mg of ALA through caudal vein, and then under red light exposure 37
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(635 nm, 450 mW) for 10 min after 4 h post injection; (Ⅳ) ALA microneedles + light group, in which the mice were first treated with ALA-loaded microneedle patches (122 μg of ALA per microneedle patch, 5 patches), and then under light exposure (635 nm,
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450mW) for 10 min after 4 h post insertion. Each microneedle patch was inserted into skin for 10 min and then removed. Each data point represents mean ±standard deviation
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(n = 4).
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Fig. 8. The skin recovery after microneedle treatment. The representative images of
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mice treated with a microneedle patch for 2min.Skin puncture marks were photographed at 0 min, 10 min, 30 min, 1 h, 3 h and 17 h post treatment.
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Xiao Zhao, Xinfang Li, Peng Zhang, Jianwei Du, Youxiang Wang PII: DOI: Reference:
S0168-3659(18)30432-2 doi:10.1016/j.jconrel.2018.07.038 COREL 9398
To appear in:
Journal of Controlled Release
Received date: Revised date: Accepted date:
2 April 2018 10 July 2018 25 July 2018
Please cite this article as: Xiao Zhao, Xinfang Li, Peng Zhang, Jianwei Du, Youxiang Wang , Tip-loaded fast-dissolving microneedle patches for photodynamic therapy of subcutaneous tumor. Corel (2018), doi:10.1016/j.jconrel.2018.07.038
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Tip-loaded fast-dissolving microneedle patches for photodynamic therapy of subcutaneous tumor Xiao Zhao#, Xinfang Li#, Peng Zhang, Jianwei Du, Youxiang Wang
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MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310027, P.R.
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China.
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Abstract
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5-Aminolevulinic acid (ALA) based photodynamic therapy (PDT) is a modality for the treatment of cancers. However, due to its hydrophilicity and zwitterionic nature, the
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transdermal delivery of ALA is limited for the PDT of subcutaneous tumor. To address this problem, tip-loaded fast-dissolving microneedles made of sodium hyaluronate (HA)
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were fabricated by two casting method. 122 μg of ALA was loaded per microneedle patch and mainly distributed in the tips, which could improve the utilization of drug and avoid the waste of drug residue in the base of microneedle patch after use. The HA
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microneedles could pierce stratum corneum with insertion depth about 200 μm in
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isolated skin. After insertion, HA microneedles were rapidly dissolved to release the encapsulated drug to improve patients’ convenience and compliance. Importantly, in a subcutaneous mouse tumor model established in BALB/c nude mice, the PDT efficacy of ALA-loaded HA microneedle group was much better than ALA injection group in spite of a relatively lower ALA dose with HA microneedles. The tumor inhibition rate of
#
X. Zhao and X. Li contributed equally to this work.
Corresponding author. E-mail address: [email protected](Y.X. Wang)
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ALA-loaded HA microneedle group (containing 0.61 mg of ALA) was up to 97%, while the tumor inhibition rate of ALA injection group (containing 1.65 mg of ALA) was just 66%. In addition, microchannels created by microneedle patch were quickly recovered
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within 3 h after insertion. Overall, the tip-loaded fast-dissolving HA microneedle patch
subcutaneous tumor in an efficient and safe manner.
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with ALA as drug was promising and might improve topical PDT efficacy of
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Key words: Photodynamic therapy; Dissolving microneedles; Subcutaneous tumor; HA
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1. Introduction
Photodynamic therapy (PDT) is a favorable method for the treatment of various
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tumors and non-neoplastic diseases[1, 2]. In PDT, a combination of the photosensitizer, oxygen and light at a fixed wavelength generates highly cytotoxic reactive oxygen species (ROS) to kill selected cells, obtaining efficient therapeutic outcome with
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minimal side effect[3]. 5-Aminolevulinic acid (ALA), a precursor of second generation
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photosensitizer, can be converted to endogenous protoporphyrin IX (PPIX) after a series of reactions in mitochondria[4]. ALA-based PDT has been widely applied for a number of skin lesions, such as actinic keratoses[5], basal cell carcinoma[6, 7] and squamous cell carcinomas[8]. Compared with systemic administration, topical administration is more favorable, since photosensitization only occurs locally at the site of treatment and avoids severe skin photosensitivity[8-10]. However, the transdermal penetration of ALA is
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limited by its physiochemical characteristics, such as hydrophilicity and zwitterionic nature[11]. To increase the transdermal delivery of ALA, plenty of chemical and physical techniques are explored, such as penetration enhancer[11], lipophilic derivatives[12],
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nanoemulsions[13], iontophoresis[14], sonophoresis[15] and microneedles[16]. In contrast to
17]
. Microneedles are usually considered as a
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performance and little side effect[9,
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other methods, microneedle technology gets a widespread attention for excellent
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synergistic combination of transdermal patch and hypodermic needle, which can pierce the protective barrier of the skin, stratum corneum, to promote the transdermal drug 19]
. Through the reversible microchannels created in the skin, the
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delivery[18,
penetrability of skin-impermeant drugs is enhanced. Furthermore, microneedle insertion
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causes minimal pain compared with injection needles, because the limited insertion depth of microneedles will not stimulate nerves in the skin[20].
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Microneedle-mediated ALA-based PDT has been studied to explore its applications for treating tumors. Donnelly et al. investigated the potential of solid
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silicon microneedles for treatment of tumor via poke-and-patch method[9]. This strategy accelerated the penetration of ALA through the murine skin and induced higher level of PPIX in vivo experiments. Recent work by Gill et al. showed ALA-coated stainless microneedles promoted the transdermal delivery of ALA and its inhibition effect on subcutaneous tumor was around 57% with a dose of just 1.75 mg of ALA, while the topical cream including 5 mg of ALA was not capable of suppressing tumor growth [17].
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Nevertheless, no matter poke-and-patch method or coated solid microneedles, these inorganic or metal microneedles suffer from some issues. The microchannels created by microneedles in poke-and-patch method reseal quickly after removing microneedle patches, which precludes the further penetration of the encapsulated drug[18]. The drug 21]
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loading of microneedles by coating or dipping is low[20,
. In addition, inorganic
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microneedles are brittle to rupture in the skin, which may cause safety issues[22]. Mental
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microneedles must be removed and threw away as sharp biohazardous waste after use[23].
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Therefore, a kind of microneedle patch with sufficient mechanical strength, safety, easy
microneedle patch is one of them.
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fabrication and relatively larger drug loading capacity is needed, and the polymer
In recent years, polymer microneedles have been widely used to transdermally
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deliver a variety of drugs, especially hydrophilic drugs and macromolecular drugs[24, 25]. To date, many polymer materials have been utilized to fabricate polymer microneedles, such as sodium hyaluronate (HA)[26,
27]
, chitosan[22,
28]
, polyvinyl alcohol[29],
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polyvinylpyrrolidone[30, 31] and polylactic acid[32]. Polymer microneedles have sufficient
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mechanical strength to puncture the stratum corneum and maintain the stability of drugs. During application, although some polymer microneedles may rupture in the skin, it is safe because of good biocompatibility and biodegradability of polymer materials[21, 30]. HA is a sort of biocompatible polysaccharide and an important ingredient of the skin, which has been approved by the U.S. Food and Drug Administration (FDA) to fill soft tissue damage in 2003. HA can promote cell movement and proliferation, inhibit cell
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differentiation and regulate the conglutination of cell and extracellular matrix. And the degradation product, monosaccharide, can also take part in metabolism circulation. In this study, we utilized HA to fabricate tip-loaded fast-dissolving polymer microneedle
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patch to enhance the transdermal delivery of ALA for PDT of subcutaneous tumor. The basic properties of HA microneedles, such as mechanical strength, insertion depth,
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dissolution behavior and drug release were investigated systematically. Next, in vivo
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PDT efficacy of ALA-loaded HA microneedle patch was examined in a subcutaneous
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mouse tumor model using human oral epidermoid carcinoma (KB) cells. Finally, skin recovery was simply tested in BALB/c nude mice. The tip-loaded fast-dissolving HA
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microneedle patch could adjust drug loading efficiency easily and might improve
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2. Materials and methods
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topical PDT efficacy of subcutaneous tumor in an efficient and safe manner.
2.1 Chemicals
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Sodium hyaluronate (HA, Mw approximately 10kDa) was supplied by Zhenjiang Dong Yuan Biotech Co., Ltd. (Zhenjiang, China). Rhodamine 6G, 5-aminolevulinic acid
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(ALA), sodium sulfite, and 2,4,6-picrylsulfonic acid solution (TNBS, 5% w/v) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Sodium dihydrogen phosphate dihydrate was purchased Huzhou Hushi Chemical Reagent Co., Ltd. (Huzhou, China). Polydimethylsiloxane (PDMS, Sylgard 184) and the optimum cutting temperature (OCT) compound were purchased from Dow Corning (Wiesbaden, Germany) and Sakura Finetek Japan Co., Ltd. (Hamacho, Japan), respectively. PDMS
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molds of microneedles (with about 1000 μm height, 300 μm width at base, 1500 μm needle pitch and 5×5 array) were purchased from Micropoint Technologies Pte. Ltd. (Singapore). Ultra-pure deionized water (Millipore, Direct Q® 3 UV) was used for all
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the experiments. All other chemicals were of analytical grade. 2.2 Cell culture
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Human oral epidermoid carcinoma (KB) cells were provided by China Centre for
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Typical Culture Collection and cultured in RPMI 1640 medium containing 10%
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heat-inactivated fetal bovine serum, 1% penicillin and 1% streptomycin under 5% CO2 at 37 ℃. All experiments were conducted in the logarithmic phase of cell growth.
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2.3 Animals
All animal experiments were carried out according to the “Principles of Laboratory
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Animal Care” (NIH publication no. 86-23, revised 1985) and the guidelines for Animal Care and Use Committee, Zhejiang University. Healthy male Sprague-Dawley rats (SD
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rats, 6 weeks old, weight around 200 g) and male BALB/c nude mice (5 weeks old, weight around 15 g) were supplied by the animal center of Zhejiang Academy of
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Medical Sciences and Shanghai SLAC Laboratory Animal Co., Ltd., respectively. The abdominal skin of Sprague-Dawley rat (SD rat) with hair shaved was pre-prepared and kept in the -80 ℃ super cold refrigerator for in vitro skin insertion tests. To set up the tumor model, KB cells in PBS buffer (pH7.4) were injected (100 μL, 2.2 × 106 cells/mouse) subcutaneously into the right flank of BALB/c nude mice. 2.4 Fabrication of tip-loaded fast-dissolving HA microneedle patch
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The tip-loaded fast-dissolving HA microneedles were fabricated via two-casting method (Fig. 1)[20,
33]
. Briefly, PDMS inserts in 50 mL corning tubes were first
manufactured. Next, drug-loaded casting solution containing 1 g of HA, 92.8 mg of
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ALA and 2 mL of deionized water were prepared. Following that, 50 μL of the prepared drug-loaded solution was poured into each PDMS mold and then placed in 50 mL
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corning tubes containing PDMS inserts. After that, they were centrifuged (Heraeus
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Biofuge Stratos Centrifuge, Thermo scientific, USA) in a swinging bucket rotor at 4,500
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rpm for 25 min to fill the cavities of the PDMS mold and form the tips of microneedles. Superfluous solution left on the surface of PDMS molds was removed by pipette. The
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molds were re-centrifuged for 3 min to promote compaction. A pure HA gel solution (50 wt.%) was added to the surface of the molds and centrifuged at 4,500 rpm for 45min to
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form the base of microneedles. Then, these molds were dried in an oven at 37 ℃ overnight. Finally, the ALA-loaded HA microneedle patches were gently detached from
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the molds and kept in the desiccator containing silica gel. The rhodamine 6G-loaded HA microneedle patches were fabricated by the same production process. The microneedle
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patches were stored in the dry and dark environment for further use. 2.5 Morphology and drug loading of microneedles The morphology of ALA-loaded HA microneedles was examined by a stereoscope (VHX-2000, Keyence, Japan). The sizes of microneedles were analyzed by Image J. Details of dimensions were illustrated, including the height, tip diameter, width of base and needle pitch of ALA-loaded HA microneedles. To observe the distribution of drug
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in microneedle patches, rhodamine 6G, a red fluorescent dye, was used as a model drug. The ALA-loading content in the HA microneedle patch was determined using TNBS method according to a previously reported literature[34]. In brief, each
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ALA-loaded HA microneedle patch was fully dissolved in 1mL of deionized water to release all the encapsulated ALA, then the solution was centrifuged at 12000 rpm for 5
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min to collect the supernatant. 600 μL of mixed solution with equal volume of obtained
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supernatant, borate buffer (0.1 mol/L, pH8.4), and TNBS solution (0.1% w/v) was
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reacted at 50 ℃ for 40 min, then 200 μL of sodium dihydrogen phosphate solution (0.1mol/L) containing 2×10-6 mol of sodium sulfite was added to the mixed solution to
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stop the reaction. After that, the mixed solution was kept at room temperature for 20 min, and ultraviolet-visible spectrophotometry (UV-2550, Shimadzu, Japan) was
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utilized to analyze the mass of released ALA according to the standard curve. 2.6 Mechanical property of microneedles
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A universal testing machine (5543A, Instron, USA) was used to preliminarily assess the mechanical property of ALA-loaded HA microneedles as previously
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reported[28]. Each ALA-loaded HA microneedles was put on a stainless steel plate with tips upward. The force, perpendicular to the plate, was applied by a moving sensor to press ALA-loaded microneedles at the constant speed of 200 μm/s. The machine recorded the force of moving sensor as a function of ALA-loaded HA microneedles displacement. In addition, initial and final morphology of ALA-loaded HA microneedles during compression test were obtained using a stereoscope.
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2.7 In vitro skin insertion tests 2.7.1 In vitro skin insertion ability of microneedles To further estimate the strength of microneedles, the isolated abdominal skins of
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SD rats were first taken out from refrigerator and soaked in 0.9 wt.% sodium chloride solution to reach the room temperature, then the water on the skin surface was sucked
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by filter papers. Next, these skins were fixed on a foam board before the experiment.
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Following that, the rhodamine 6G-loaded HA microneedle patch was inserted into the
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skins using a thumb for 2min. After insertion, the rhodamine 6G-loaded HA microneedle patch was removed. The skin surface was rubbed carefully by presoaked
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cotton swab to make sure that no rhodamine 6G was remained on the skin surface. Then these skins were examined by a digital camera to calculate the insertion ratio, which
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was calculated by dividing the number of remained red dots on the skin surface by the total number of needles of microneedles. Finally, the skin was excised to determine the
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insertion depth by frozen tissue sections. And the insertion depth was defined as the average depth of holes created by the microneedle patch.
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The samples of frozen tissue sections were prepared as follows. Microneedle insertion area was separated from the skin with a scalpel. The isolated skin sections were put into -80 ℃super cold refrigerator for about 2 min, then the sample was embedded in OCT compound in a cryostat mold. The frozen samples were cut into 10 μm thick sections using a cryotome (CryoStar NX50, Thermo scientific, USA) and then placed on adhesion microscope slides (Citotest Labware Manufacturing Co., Ltd).
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Finally, the skin sections were photographed under a microscope (BX61, Olympus, Japan). Meanwhile, to characterize the permeation of drug in the vertical direction of
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full-thickness skin, the rhodamine6G-loaded HA microneedle patch was inserted into the abdominal skins of SD rats for 2 min as described above. After that, the skin was put
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on a microscope slide and analyzed with a confocal laser scanning microscope (TS SP5,
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Leica, Germany) at the excitation wavelength of 526 nm. Pictures were collected in the
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xy-plane. Scanning was operated once at the interval of 10 μm from skin surface through the vertical direction of xy-plane, and the starting position of scanning of the
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skin surface (z= 0 μm) was viewed as the plane with the brightest fluorescence. 2.7.2 In vitro dissolution of microneedles
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The dissolution rate of the ALA-loaded HA microneedle patch was investigated on the isolated abdominal skins of SD rats. The SD skins were pretreated as described
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above, then ALA-loaded HA microneedle patches were applied on the skins using a thumb for different time. At the indicated time points, ALA-loaded HA microneedle
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patches were removed from the skin and observed by a stereoscope. And the size variations of microneedles were analyzed by Image J. 2.7.3 In vitro transdermal delivery of drug In vitro release of ALA from ALA-loaded HA microneedle patches was investigated by Franz diffusion cell (RYJ-12B, Huang Hai Yao Jian, China) as previous report[35, 36]. The isolated SD skins were first pretreated as described above. ALA-loaded
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HA microneedle patch was then inserted into the skin using a thumb for 2 min and fixed to the skin by medical adhesive tape. Next, the skin with microneedle patch was mounted onto the Franz cell with dermal side facing the receptor chamber. The permeation area of Franz cell was about 2.39 cm2. The receptor medium (7.1 mL of
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PBS, pH7.4) was maintained 32 ℃in an incubator and continuously stirred 300 rpm. At
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predetermined intervals, samples (0.6 ml) were withdrawn from the receptor chamber
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and replaced with an equal volume of fresh PBS buffer. Subsequently, the samples
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extracted from cells were stored at -20 ℃ until analysis. The cumulative permeation amount of ALA from microneedles was determined using TNBS method as discussed
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above. 2.8 In vivo experiments
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2.8.1 Antitumor efficacy of microneedles To evaluate the photodynamic therapeutic effect of ALA-loaded microneedle patch
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in vivo, treatment was begun when the tumor volume reached around 130 mm3. Tumor bearing mice were randomly divided into four groups (n=4 per group): (1) Untreated
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group, in which the mice were untreated; (2) Only light group, in which the mice were just treated with the light exposure (635 nm, 450 mW) for 10 min; (3) ALA injection + light group, in which the mice were first injected 100 μL of heat-inactivated PBS buffer (pH 7.4) containing 1.65 mg of ALA through caudal vein, and then under light exposure (635 nm, 450 mW) for 10 min after 4 h post injection; (4) ALA microneedles + light group, in which the mice were first treated with ALA-loaded microneedle patches (122
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μg of ALA per microneedle patch, 5 patches), and then under light exposure (635 nm, 450mW) for 10 min after 4 h post insertion. Each microneedle patch was inserted into skin for 10 min and then removed. The therapeutic effect was examined by the change of tumor volume. Tumor sizes were measured with an electronic digital caliper, and
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tumor volume was calculated using the formula: tumor volume = 0.5 × length ×
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width2[37]. After 14 d therapy, tumor bearing mice were sacrificed to obtain the images
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and weights of tumors of each group to accurately estimate the therapeutic efficacy, and
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the tumor inhibition rate was calculated by dividing the excised tumor volume of the treatment group by the excised tumor volume of the control group.
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2.8.2 Skin recovery after microneedle insertion
Skin recovery ability after microneedle insertion was simply evaluated on the skin
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of BALB/c nude mice in vivo. Briefly, the ALA-loaded HA microneedle patch was inserted into the back skin of BALB/c nude mice for 2 min, then the microneedle patch
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was removed from the skin surface. Images of the insertion area after microneedle treatment were obtained by a digital camera at 0 min, 10 min, 30 min, 1 h, 3 h, and 17 h
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after insertion.
2.9 Statistical analysis For this study, all values were presented as their mean ± standard deviation. Statistical differences were considered to be significant when p < 0.05. 3. Results and discussion 3.1 Morphology and drug loading of microneedles
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To prepare the tip-loaded fast-dissolving HA microneedles, a two-casting fabrication process was adopted. As shown in the Fig. 1, the mixed HA solution containing ALA was first poured into PDMS molds to form the tips of microneedles,
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then the blank HA gel solution without ALA was cast to PDMS molds to form the base of microneedles.
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As shown in the Fig. 2, the fabricated microneedles had the identical structure of
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pyramidal shape. ALA-loaded fast-dissolving HA microneedles consisted of 25 (5×5)
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pyramidal needles in Fig. 2(a). The height, tip diameter, width of base and needle pitch were 907 + 20 μm, 12 + 5 μm, 309 + 16 μm and 1123 + 2 μm, respectively. It was
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approximately 9.3% reduction in height compared to PDMS mold, which might be caused by water evaporation during drying process[38]. And the base area was a thick
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and rigid board. Thus the force acting on the baseplate could be uniform to facilitate the microneedles insert into the skin by hand both in vitro and vivo. Meanwhile, rhodamine
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6G was chosen as model drug to visualize the distribution of drug in the microneedles. As shown in Fig. 2(b), although the color of the base of rhodamine 6G-loaded HA
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microneedles was slightly deepened, rhodamine 6G was mainly dispersed in the tips of microneedles. It indicated this design, tip-loaded microneedle patch, could improve the utilization of drug and avoid the waste of drug residues in the base of microneedle patch after treatment, and highlight the unique advantage of two-casting method to prepare microneedles. It was worth of noting that the color of the base might be attributed to the diffusion of rhodamine 6G during preparation. And ALA content in ALA-loaded HA
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microneedles was determined by TNBS method. The loading amount of ALA was 122+8 μg per microneedle patch. 3.2 Mechanical property of microneedles
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It’s necessary for polymer microneedles to have sufficient mechanical strength to pierce the stratum corneum. Factors, such as tip diameter, geometry, material
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composition and aspect ratio, may affect the strength of polymer microneedles[38, 39].
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Pyramidal microneedles were more robust than conical microneedles due to the unique
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property to the larger cross-sectional area of pyramidal microneedles at the same base width or diameter[28]. Therefore, pyramidal microneedles were chosen in this work.
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Results from mechanical compression test (Fig. 3a) showed that the force acting on ALA-loaded HA microneedles elevated with the increase of the displacement of
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microneedles without a distinct transition point. ALA-loaded HA microneedles (Fig. 3b) undertook progressive deformation without notable separation during compression test. The force reached 0.6 N/needle when the displacement was 0.2 mm, which was
.
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41]
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obviously comparable or even superior to performances from previous reports[28, 38, 40,
3.3 In vitro skin insertion tests 3.3.1 In vitro skin insertion ability of microneedles To further examine the strength of microneedles, in vitro insertion test was carried out to obtain the skin insertion ratio and insertion depth using the isolated abdominal skin of SD rats with hair shaved. The rhodamine 6G-loaded HA microneedle patch was
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applied to the skin for 2 min and subsequently removed from the skin. The red dots (Fig. 4a) that couldn’t be wiped out on the skin surface was rhodamine 6G left in the skin, indicating that microneedles had successfully inserted into the skin. Furthermore, the
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histological results (Fig. 4b) further verified the result, and it showed red dye had spread from the holes created by microneedles to the surrounding tissue.
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As shown in the Fig. 4(a) and Fig. 4(b), the skin insertion ratio was 100%, and the
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insertion depth was 218 + 52 μm, which was far shorter than the height of microneedles
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(approximately 907 μm). The proper reason could be ascribed to two aspects: Firstly, it might be the result of elasticity of the skin[38]. Secondly, when microneedles were
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inserted into the skin, HA could adsorb physiological fluid and the tips dissolved quickly. The insertion process accompanied with dissolution. Thus the insertion depth
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was much shorter than the height of microneedles. The microneedles could pierce through the stratum corneum (the thickness was10-20 μm) and reach the dermis layer
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without piercing through the skin.
Meanwhile, in order to determine the penetration depth of encapsulated drugs in
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the skin, the skin treated by rhodamine 6G-loaded HA microneedle patch was observed using a confocal microscope. The skin insertion site was scanned at varying depth in the direction perpendicular to the skin surface. As shown in the Fig. 4(c), after insertion for 2 min, the penetration depth of rhodamine 6G could reach 600 μm. The histological section in Fig. 4(b) suggested the insertion depth was only 218±52μm. So in the Fig. 4(c), we could see red fluorescence from the surface to the depth of 200μm, which was
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the position of tips actually. On the other hand, the microneedles had the identical structure of pyramidal shape in this research. The drug content near the base area of the microneedles was much larger than that in tips. It was normal to observe that the red
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fluorescence intensity at the depth of 200μm was less than that near surface. Furthermore, red fluorescence could rapidly spread from application site to surrounding
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tissue. All results indicated the microneedles had efficient strength to perforate the
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stratum corneum and delivered the encapsulated drugs directly into dermal tissue.
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3.3.2 In vitro dissolution of microneedles
HA was commonly used as the matrix material of dissolving microneedles owing
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to its high hydrophilicity and biocompatibility[20,
41]
. The dissolution behavior of
ALA-loaded HA microneedle patch was investigated. As shown in Fig. 5, the
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dissolution rate was nonlinear. In the first 1 min, the height of microneedles was reduced by approximately 42%, indicating a fast initial dissolution. After skin insertion
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for 4min, the height of microneedles was reduced by approximately 75%. Subsequently, the dissolution rate of microneedles became relatively slow. Approximately 80% of their
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initial height was reduced after insertion for 6 min. According to Fig. 2(b), this part contained the majority of drug. It was worth noting that the dissolution height was higher than insertion depth. As we mentioned ahead, HA could adsorb physiological fluid and the tips dissolved quickly. The insertion process accompanied with dissolution. So the actual dissolution part of microneedles was obviously longer than the penetration depth. These results demonstrated that ALA-loaded fast-dissolving microneedles were
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successfully constructed, and it could reduce the treatment time and improve patients’ convenience and compliance. In the following tumor growth inhibition experiment, we chose insertion time for 10 min to fully release the encapsulated drug.
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3.3.3 In vitro transdermal delivery of drug Drug release behavior of microneedles in vitro was examined by Franz diffusion
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cell. ALA-loaded HA microneedles were inserted into the isolated abdominal skin of SD
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rats, and drug release was monitored for 4 h. As shown in Fig. 6, in the first 5 min, the
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release of ALA from HA microneedles exhibited a burst release. HA microneedles were rapidly dissolved to release the encapsulated ALA, and the percentage of cumulative
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release was about 40% within the first 5 min. After that, the release of ALA exhibited a slow release over time. After 60 min, almost all of the encapsulated ALA was released,
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which was consistent with the published literature[41]. In vitro skin insertion tests demonstrated that the microneedles had efficient
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strength to pierce the stratum corneum，dissolved quickly, released the encapsulated drug to promote the transdermal drug delivery.
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3.4 In vivo experiments
3.4.1 Antitumor efficacy of microneedles To evaluate the therapeutic efficacy of ALA-loaded HA microneedles, tumor model was established in BALB/c nude mice using KB cells. When the tumor volume of mice grew to approximately 130 mm3, the mice were divided into four groups, and labeled as the “Untreated” group, the “Only light” group, the “ALA injection + light” group and
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the “ALA microneedles + light” group, respectively. The “Untreated” group was served as the control group. The therapeutic efficacy of PDT using subcutaneous tumor was assessed by the
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tumor volume change[42]. As shown in Fig. 7(a), the tumor volume of the control group increased rapidly, and the tumor volume was about 455% of the initial volume at day 7.
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Compared with the control group, the “Only light” group could slow tumor growth to
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certain extent. The tumor volume was about 355% of the initial volume at day 7, which
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indicated the effect of light was relatively limited. The therapeutic efficacy of the “ALA microneedles + light” group was the best, and tumor size of mice presented an obvious
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decrease trend. The tumor was about 44% of initial tumor volume in the “ALA microneedles + light” group after treatment for 7 d. Given that the tumor might recur,
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the tumor-bearing mice were examined for another week without any further treatment. After 2 weeks’ treatment, in the “ALA microneedles + light” group, the tumor of only
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one mouse relapsed in the second week while the tumor of three others remained invisible. Representative images of tumor-bearing mice of different groups at day 14
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were shown in Fig. 7(b).
After 2 weeks’ treatment, the mice were sacrificed to obtain the images and tumor weights s of each group to accurately estimate the therapeutic efficacy. As shown in Fig. 7(c) and Fig. 7(d), tumor grew most rapidly in the control group, and the average weight of excised tumor was about 1.65 g. In the “Only light” group, the final inhibition efficacy was about 50%, it might be attributed to high light intensity (635nm, 450mW,
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10 min). In the “ALA injection + light” group (containing 1.65 mg of ALA), its final inhibition efficacy was about 66%, slightly higher than only light group. However, the final inhibition efficacy of the “ALA microneedles + light” group (containing 0.61 mg
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of ALA) was the highest up to 97%. The PDT efficacy of ALA-loaded HA microneedles group was better than ALA
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injection group in spite of a lower ALA dose (microneedles: 0.61 mg vs injection:1.65
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mg ALA). It might be the following reasons. In the case of injection group, ALA
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injected through caudal vein would circulate around the body before reaching the tumor tissue, diminishing the conversion rate of ALA into PPIX. Therefore, it would be
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difficult to exploit the drug to its fullest. Instead, ALA-loaded HA microneedle patches were directly applied to the skin over the subcutaneous tumor in situ, which would have
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higher utilization of drugs and improve the therapeutic efficacy. 3.4.2 Skin recovery after microneedle insertion
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Lastly, the skin recovery rate was simply evaluated by observing the surface appearance of skin damage. The ALA-loaded microneedle patch was applied to the back
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skin of BALB/c nude mice in vivo and subsequently removed 2 min later. After treatment, there were no obvious side effect except for some visible micro-pores and local erythema in insertion site (Fig. 8). After 3 h post insertion, the majority of erythema rapidly faded away, and microchannels created by microneedles were almost resealed. Resealed microchannels were capable of reducing the risk of infection and entry of pathogens[18,
21]
. What’s more, the insertion skin reverted to its original
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condition after 17 h post insertion, indicating that the skin had good recovering ability after microneedle treatment. In vivo PDT and skin recovery experiment demonstrated the tip-loaded
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fast-dissolving HA microneedle patch was an efficient and safe way for transdermal drug delivery. However, considering the differences between mice skin and human skin,
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further experiments still need to be conducted systematically to prepare for clinical
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studies in the future.
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Conclusion
This study demonstrated that the tip-loaded fast-dissolving HA microneedle patch
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with ALA was an efficient and safe way for PDT of subcutaneous tumor. Tip-loaded HA microneedles were fabricated by two-casting method. Each microneedle patch contained
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122 μg of ALA, which were mainly distributed in the tips of needles. The insertion ratio and insertion depth were 100% and approximately 200 μm, respectively. After skin
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insertion for 4 min, the dissolved height percentage of microneedles was about 75%. These results suggested the microneedles had efficient strength to pierce the stratum
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corneum，dissolved quickly, released the encapsulated drug to promote the transdermal drug delivery. In the subcutaneous mouse tumor model, the PDT efficacy of ALA-loaded HA microneedles group was better than ALA injection group. The tumor inhibition rate of ALA-loaded HA microneedles group (containing 0.61 mg of ALA) was up to 97%, while the tumor inhibition rate of ALA injection group (containing 1.65 mg of ALA) was just 66%. In addition, microneedle insertion caused no obvious side
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effect, and the skin had good recovering ability after microneedle treatment. Therefore, our study suggested that the tip-loaded fast-dissolving HA microneedle patch with ALA was promising and might improve topical PDT efficacy of subcutaneous tumor in an
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continuous production capacity still remained a challenge.
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efficient and safe manner. Considering the potential application in clinical, the
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Acknowledgements
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This work was financially supported by the Natural Science Foundation of Zhejiang Province (No. LY18E03001)， Zhejiang science and technology program
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(2016C04002) and the National Natural Science Foundation of China (No. 21474087).
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Figure Captions Fig.1 Schematic illustration of the process to fabricate the tip-loaded fast-dissolving HA
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microneedle patch.
Fig.2 Stereomicroscopy images of ALA-loaded fast-dissolving HA microneedle patch
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(a) and rhodamine 6G-loaded fast-dissolving HA microneedle patch (b). Diagrammatic
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representation of the microneedle patch and its geometric parameters (c).
Fig. 3 Mechanical behavior of ALA-loaded HA microneedle patch. (a) Force was
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measured as a function of microneedles displacement, and the inset illustrates the schematic diagram of the measuring device. (b) Initial and final images of ALA-loaded
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HA microneedles were obtained during compression test. Each data point represents
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mean ± standard deviation (n = 3).
Fig. 4 Skin insertion ability of rhodamine 6G-loaded HA microneedle patch.
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Bright-field micrograph (a), histological section (b) and CLSM images (c) of the mouse skin were photographed after microneedle insertion for 2 min(white scale bar: 500 μm).
Fig. 5 Dissolution of ALA-loaded HA microneedle patches after their application to the skin in vitro. Dissolution data (a) and images (b) of ALA-loaded HA microneedle patches after insertion. Each data point represents mean ± standard deviation (n = 6).
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Fig. 6In vitro drug release behavior of ALA-loaded HA microneedle patches. Schematic diagram of Franz diffusion cell used (a). In vitro drug release profiles (b). The partial
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enlarged drawing of the dotted box (c). The loading amount of ALA was about 122 μg
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per patch. Each data point represents mean ± standard deviation (n = 4).
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Fig. 7 Photodynamic therapy of subcutaneous tumor. (a) Tumor growth curves of
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different groups of tumor-bearing mice after treatment. (b) Representative images of different groups of tumor-bearing mice after 14 days’ treatment. (c) Weight of excised
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tumor of each group after 14 days’ treatment. (d) Images of excised tumor of each group after 14 days’ treatment. Treatment was started when the tumor volume reached the size
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of about 130 mm3. Tumor bearing mice were randomly divided into four groups: (Ⅰ) Untreated group, in which the mice were untreated; (Ⅱ) Only light group, in which the mice were just treated with the light exposure (635 nm, 450 mW) for 10 min; (Ⅲ) ALA
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injection + light group, in which the mice were first injected 100 μ L of
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heat-inactivated PBS containing 1.65 mg of ALA through caudal vein, and then under red light exposure (635 nm, 450 mW) for 10 min after 4 h post injection; (Ⅳ) ALA microneedles + light group, in which the mice were first treated with ALA-loaded microneedle patches (122 μg of ALA per microneedle patch, 5 patches), and then under light exposure (635 nm, 450mW) for 10 min after 4 h post insertion. Each
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microneedle patch was inserted into skin for 10 min and then removed. Each data point represents mean ±standard deviation (n = 4).
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Fig. 8 The skin recovery after microneedle treatment. The representative images of mice
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min, 10 min, 30 min, 1 h, 3 h and 17 h post treatment.
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treated with a microneedle patch for 2min. Skin puncture marks were photographed at 0
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Fig. 1. Schematic illustration of the process to fabricate the tip-loaded fast-dissolving
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HA microneedle patch.
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Fig. 2. Stereomicroscopy images of ALA-loaded fast-dissolving HA microneedle patch (a) and rhodamine 6G-loaded fast-dissolvingHA microneedle patch (b). Diagrammatic representation of the microneedle patch and its geometric parameters (c).
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Fig. 3. Mechanical behavior of ALA-loaded HA microneedle patch. (a) Force was measured as a function of microneedles displacement, and the inset illustrates the
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schematic diagram of the measuring device. (b) Initial and final images of ALA-loaded HA microneedles were obtained during compression test. Each data point represents
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mean ± standard deviation (n = 3).
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Fig. 4. Skin insertion ability of rhodamine 6G-loaded HA microneedle patch. (a)Bright-field micrograph, (b) histological section and (c) CLSM images (scale bar:
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500 μm) of the mouse skin were photographed after microneedle insertion for 2 min.
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Fig. 5. Dissolution of ALA-loaded HA microneedle patches after their application to the
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skin in vitro. Dissolution data (a) and images (b) of ALA-loaded HA microneedle
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patches after insertion. Each data point represents mean ± standard deviation (n = 6).
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Fig. 6.In vitro drug release behavior of ALA-loaded HA microneedle patches. Schematic diagram of Franz diffusion cell used (a). In vitro drug release profiles (b).
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The partial enlarged drawing of the dotted box (c). The loading amount of ALA was
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about 122 μg per patch.Each data point represents mean ± standard deviation (n = 4).
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Fig. 7. Photodynamic therapy of subcutaneous tumor. (a) Tumor growth curves of different groups of tumor-bearing mice after treatment. (b) Representative images of
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different groups oftumor-bearing mice after 14 days’ treatment. (c) Weight of excised
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tumor of each group after 14 days’ treatment. (d) Images of excised tumor of each group after 14 days’ treatment. Treatment was started when the tumor volume reached the size of about 130 mm3. Tumor bearing mice were randomly divided into four groups: (Ⅰ) Untreated group, in which the mice were untreated; (Ⅱ) Only light group, in which the mice were just treated with the light exposure (635 nm, 450 mW) for 10 min; (Ⅲ) ALA injection + light group, in which the mice were first injected 100 μL of heat-inactivated PBS containing 1.65 mg of ALA through caudal vein, and then under red light exposure 37
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(635 nm, 450 mW) for 10 min after 4 h post injection; (Ⅳ) ALA microneedles + light group, in which the mice were first treated with ALA-loaded microneedle patches (122 μg of ALA per microneedle patch, 5 patches), and then under light exposure (635 nm,
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450mW) for 10 min after 4 h post insertion. Each microneedle patch was inserted into skin for 10 min and then removed. Each data point represents mean ±standard deviation
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(n = 4).
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Fig. 8. The skin recovery after microneedle treatment. The representative images of
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mice treated with a microneedle patch for 2min.Skin puncture marks were photographed at 0 min, 10 min, 30 min, 1 h, 3 h and 17 h post treatment.
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Figure 1
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