Transdermal Delivery of Living and Biofunctional Probiotics through

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Transdermal Delivery of Living and Biofunctional Probiotics through Dissolvable Microneedle Patches Hui-Jiuan Chen, Di-an Lin, Fanmao Liu, Lingfei Zhou, Di Liu, Zhihong Lin, Chengduan Yang, Quanchang Jin, Tian Hang, Gen He, and Xi Xie ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00102 • Publication Date (Web): 04 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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ACS Applied Bio Materials

Technologies, School of Electronics and Information Technology, Sun YatSen University

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Transdermal Delivery of Living and Biofunctional Probiotics through Dissolvable Microneedle Patches Hui-Jiuan Chen1+, Di-an Lin1+, Fanmao Liu1, Lingfei Zhou1, Di Liu,2 Zhihong Lin1, Chengduan Yang1, Quanchang Jin1, Tian Hang1, Gen He1,*, Xi Xie1,* 1

The First Affiliated Hospital of Sun Yat-Sen University; State Key Laboratory of

Optoelectronic Materials and Technologies, School of Electronics and Information Technology, Sun Yat-Sen University, Guangzhou, China 2

Pritzker School of Medicine, University of Chicago, IL, USA, 60637

+

These authors contributed equally to this work.

*To whom correspondence may be addressed. Corresponding to: Xi Xie, [email protected]; Gen He, [email protected]

Abstract Bioactive functional probiotics play an important role in many health applications such as maintaining the skin health and the immunity of human host. Artificial supplementation of probiotics would enhance immune functions as well as regulating skin health. However, simple and effective methods to deliver probiotics into the dermis to regulate local dermal tissue are still lacking. Furthermore, microneedles have been used for transdermal drug delivery in a pain-free manner, yet there were rare reported methods to deliver living microbes via microneedles. In this work, we developed a technique to deliver bioactive functional probiotics, using lactobacillus as the model probiotic, into local dermis by dissolvable microneedles. The transdermal delivery of probiotics might enhance local skin regulation and immunity, and dissolvable microneedles served as a safe and pain-free tool for dermal microbial delivery. Lactobacillus were encapsulated in dissolvable microneedles with high viability by centrifugation casting method. The microneedles rapidly dissolved after skin penetration, releasing the lactobacillus into the subcutaneous space, without


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causing local tissue irritation. The lactobacillus were functionally bioactive following transdermal delivery, actively synthesizing lactic acid both ex vivo and in vivo. Our technique provided a safe, effective, and convenient approach for the transdermal delivery of probiotics into local skin, with the potential to improve skin health and immunity.

Keywords ： dissolvable microneedle, transdermal delivery, bioactive probiotics delivery, probiotics stability, transdermal delivery efficiency Introduction Natural probiotics are living microorganisms that are beneficial to the human host, and they play an important role in digestion, skin health, host metabolism, anti-inflammation, and immunity for cancer.1–4 For example, lactic acid-producing bacteria exhibited antioxidant abilities that improved the host’s immune regulation and prolonged the host’s life span.5–8 In particular, skin microbes on the epidermis and dermis play an important role in maintaining skin health and protects the host against pathogen invasion.9,10 Moreover, probiotics can produce large amounts of organic acid, such as lactic acid, to reduce the pH of the skin or digestive tract to inhibit the growth of pathogenic bacteria and to improve immunity.11 However, daily applications of antibiotics and antimicrobials eliminate the entire microbiome and lacks specificity for pathogenic microbes and prevent the establishment of a healthy microbiota.12,13 Furthermore, probiotics are often used to enhance immunity, to regulate skin health, to inhibit tumor growth, and to slow aging.14,15 Probiotics are generally provided as oral supplements, but many probiotics strains do not survive along the digestive tract, and the oral ingestion of probiotics are ineffective for localized applications such as regulating skin health. Topical applications of probiotics have been reported to improve the skin surface health,16,17 but the stratum corneum is impermeable to probiotics and serve as a barrier for dermal delivery. Transdermal injection with metal needles is capable of delivering micro-objects into subcutaneous



tissue, but metal needle injection tends to produce pain and undesirable injury and infection risks. In addition, the successful delivery of probiotics requires a stable package of probiotics without affecting their viability.18–20 Therefore, the transdermal delivery of probiotics for local skin treatment has not been explored. Microneedle (MN) is a recently developed method of noninvasive or minimally invasive route to transdermally deliver targeted drugs.21 MN administration penetrates the skin’s stratum corneum to release loaded drugs directly into the subcutaneous space, without pricking the nerves to cause pain.22 The first generation of MN arrays were made of silicon, metals or organic polymers, and were designed to create micro-pores on the skin which allows the loaded drugs to diffuse into the skin.23,24 More recently, dissolvable materials were employed to fabricate MNs, which could dissolve rapidly after penetrating the skin to release drugs.25–28 Dissolvable MNs have been reported to effectively deliver vaccine, analgesics, anticatarrhals, insulin, and anti-obesity medications.29–40 In spite of these successes, MNs are most widely used to delivery pharmaceutical agents, and the delivery of living microbes or biological cells has rarely been reported, to the best of our knowledge. The successful delivery of living microbes or cells using MNs would introduce new opportunities to regulate the local skin or tissue and introduces functionalities other than pharmaceutical delivery. In this work, we reported the successful transdermal delivery of bioactive probiotics into the dermis of the local skin using dissolvable MNs, Figure 1. The transdermal delivery of probiotics can potentially improve local skin regulation as well as immunity, and dissolvable MNs enables safe, pain-free, and repeated applications of microbes. Lactobacillus (LABs), one of the most common probiotics, were employed as the model microbe to demonstrate delivery through MNs. Sodium carboxymethyl cellulose (SCMC) MN arrays loaded with LABs (LABs-MN) were fabricated by a centrifugation casting method using an inverted cone-shaped mold, without reducing LABs viability and proliferation. The SCMC MNs rapidly dissolved following skin penetration, releasing the LABs into the subcutaneous space, without inducing local tissue irritation. The delivered LABs actively synthesized lactic acid in ACS Paragon Plus Environment

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the pigskin as well as in living rats, demonstrating that the delivered LABs were functionally bioactive in vivo. This study provided a safe, effective, and convenient approach to supplement probiotics into local skin and might open new opportunities to improve local skin health and immunity.

Figure 1. Schematic of dissolvable MN patch mediating transdermal delivery of living and biofunctional probiotics. The SCMC MNs rapidly dissolved following skin penetration, releasing the bioactive LABs into the subcutaneous space.



Results and Discussion

Figure 2. (a) Illustration of the fabrication procedure of the MN array loaded with LABs. The LABs-MN was fabricated by a centrifugation casting method using an inverted cone-shaped mold. (b) Characterization of the MN arrays. (b-1) photographic image of the MN array patch; (b-2) photographic and SEM images of the MN array. (b-3) photographic and SEM images of the dissolved MN array. Dissolvable MNs were investigated as pain-free delivery tool for probiotics, and highly stable probiotic encapsulation along with the rapid release of probiotics was achieved following skin penetration. LABs is one of the most common probiotics and it actively synthesizes lactic acid in the body to maintain health. LABs can aid in the treatment of various diseases including vaginal infection and skin disorders. Here, LABs were employed as the model probiotic to demonstrate delivery via MNs. The MNs were fabricated by a centrifugation casting approach using an inverted cone-shaped mold, Figure 2a. Briefly, 3D MN-shaped geometry of the negative photoresist, SU-8, was fabricated via inclined/rotated ultraviolet (UV) lithography to create sharp V-shaped tips at the end of needles for penetration, Figure 2-a1. In the ACS Paragon Plus Environment

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second step, inverted cone-shaped mold made of polydimethylsiloxane (PDMS) was generated from the initial SU-8 positive MN mold through injecting molding method, Figure 2-a2. PDMS solution in a 10:1 (by volume) ratio of elastomer to its curing agent was poured over the SU-8 MN array mold. After curing at 60 oC for 24 h, the resultant PDMS molds were peeled off from the SU-8 MN array, Figure 2-a3. After fabricating the molds, dissolvable MNs containing LABs were prepared through centrifugation casting method. SCMC solution containing LABs was added into the PDMS mold and driven into the mold cavities through centrifugation, followed by drying at 40 oC for 2 h to form solid SCMC MN tips with LABs localized in the tips, Figure 2-a4 and a5. After initial centrifugation, SCMC solution without LABs was added on top of the MN tips. The second centrifugation was then carried out to form the remaining fragment of the MNs and patch base, Figure 2-a6 and a7. The as-prepared MN patch was then obtained by peeling it off from the PDMS mold, Figure 2-a4-8. This generated a MN patch with a geometry identical to the original SU-8 mold. The MN array on each patch composed of 81 (9×9) needles at a density of 81 needles per 1 cm2, Figure 2-b1. The height of each MN was 600 µm and the distance between each needle base was 250 µm, with each needle presenting as a pyramid shape, Figure 2-b2. The structure of the MN array was presented in photographic and SEM images, Figure 2b. The MNs could be readily dissolved in aqueous solution in 5 mins. This allowed the rapid delivery of encapsulated materials into the skin. After the MNs are dissolved, the sharp tips vanished and the length of the entire MN was shortened and only the part that is attached to the substrate remained, as indicated in Figure 2-b3.



Figure 3. (a) SEM image of the LABs morphology. (b) Fluorescent image of the LABs labeled with Hoechst 33342 (blue fluorescence). (c) Confocal fluorescence image of Hoechst 33342-labeled LABs-MN. (d) Merged confocal fluorescence image of LABs-loaded MNs. LABs were labeled with blue fluorescence, and MN materials were labeled with red fluorescence. (e) Photographs of dye-loaded MN-patch applied on pigskin. After removing the patch, the red dye was deposited into the skin. (f-g) Fluorescence images showing LABs-MNs penetrating pigskin. The dissolved MN materials (red fluorescence) was deposited into the pigskin. The LABs (blue fluorescence) was deposited into the pigskin as well following MN insertion. (h) The LABs were released from MNs and continued to be incubated for 36 h. The amount of LABs encapsulated in MNs were determined by counting the number of released LABs by plate counting assay. N=3. (i) The storage stability of LABs loaded in MNs was determined by counting the amount of LABs released from MNs after a certain period of incubation. The LABs loaded MN arrays were stored for 7, 14, and 21 days, respectively. N=3. The morphology of LABs was observed with SEM to be short rods, with a length of approximately 2 µm and a diameter of 300 nm, Figure 3a. The LABs could be labeled with fluorescent dye, Hoechst 33342, which selectively stained LABs nuclear contents, to enable LABs tracking with fluorescent microscopy, Figure 3b. To minimize LABs wastage and to provide accurate dosing, the LABs were loaded


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within the MN tips, ensuring the maximum delivery of LABs into the skin. The MN-containing Hoechst 33342-labeled LABs was imaged with fluorescence microscopy. As shown in Figure 3c, blue-fluorescent LABs distributed within the MN tip, indicating the successful loading of LABs. In addition, red fluorescent dye, Rhodamine B, was mixed in the MN material, SCMC, to label the MN body (Supporting Information S1). The fluorescence-labeled MN was imaged with confocal fluorescence microscopy, and the image was re-constructed with 3D view. As shown in Figure 3d, the MN tips exhibited red fluorescence, while blue fluorescent LABs were loaded in the tips. To visualize skin penetration, the MN patch had tips labeled with red fluorescent dye, Rhodamine B, and encapsulated LABs were labeled with blue fluorescent dye, Hoechst 33342, and the device was pressed against pigskin. As shown in the photographic image of Figure 3e, MN patches were pressed in a vertical direction and inserted into the skin for 5 mins. After removal of the MN patch, the treated skin samples were stained with Rhodamine B red and presented epidermal compression without visible rupture of the epidermis. The array of red fluorescent Rhodamine B retained in the pigskin, suggesting the penetration of MN tips into the skin, Figure 3f. In addition, an array of red fluorescence indicated that MNs successfully penetrated pigskin, and the array of blue fluorescence exhibited in the pigskin demonstrated the delivery of Hoechst 33342-labeled LABs into the skin, Figure 3g. The MNs were immediately dissolved by the skin’s interstitial fluid following insertion, thus releasing the LABs into the epidermis. To determine the amount of LABs loaded within the MN tips, the MN tips were dissolved in DI water to release the loaded LABs, which was then counted by plate counting assay. Briefly, MNs containing different doses of LABs were prepared by mixing SCMC solution with different concentrations (10-50 mg/ml) of LABs. The MN tips were immerged in DI water, which rapidly dissolved and the encapsulated LABs were released. The released LABs in aqueous solution was then extracted and cultured for 36 hours. Typical LABs colonies that presented as white spots on the nutrient agar media were shown in Supporting Information S2. The LABs proliferated over time, this indicated that the released LABs were alive. The amount of LABs was ACS Paragon Plus Environment


determined by plate counting. Based on the result, MN fabricated based on SCMC solution with 20 mg/ml LABs contained more than 5000 LABs in the needle tips, while MN based on SCMC solution with 30 mg/ml LABs and 40 mg/ml LABs contained around 1.0×104 LABs and 1.5×104 LABs, respectively. The loaded amount of LABs were approximately linearly correlated with the concentration of the LABs solution, Figure 3h. This indicated that MNs containing different doses of LABs could be fabricated in a well-controlled manner, providing a straightforward way to regulate the delivery of LABs. The long-term stability of LABs was examined by counting the live LABs in MNs following the storage of the MN patch at 4 oC for different time periods. MNs containing 0.3 mg LABs per patch were prepared and then stored at 4 oC for 7 days, 14 days, and 21 days, respectively. The MN tips were then dissolved and the LABs in each sample were released into DI water. The number of live LABs contained within each sample was counted with plate counting assay, Figure 3i. The results showed that the counted number of live LABs in MNs were approximately 6500 immediately following fabrication. The number of live LABs were slightly reduced to approximately 66% following storage for 21 days. The reduction of living LABs number on the first 7 days was likely due to the death of a small population of LABs that were not adapted to the fabrication process or the MN environment. However, the reduction was saturated for 7-21 days, indicated that the viability of the rest population of LABs in MNs was stable following long-term storage at 4 oC. The number of live LABs were similar amongst the samples stored for 7 days, 14 days, and 21 days. This indicated that the viability of LABs in MNs was stable following long-term storage at 4 oC. This result suggested that the LABs were able to be encapsulated in MNs while maintaining high viability. This finding was critical for the development of LABs-loaded MNs without jeopardizing the application quality.


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Figure 4. (a) Schematic of the delivery of LABs by applying a LABs-MN patch. (b) Determination of lactic acid produced by the ex vivo delivery of LABs in pigskin by LABs-MNs. Lactic acid synthesized by LABs for 1 h, 2 h, or 3 h following MNs insertion. N=3. (c) the LA level of pigskin after transdermal delivery by subcutaneous injection, LABs-MNs insertion and blank MNs. Lactic acid synthesized by LABs for 3 h following the insertion. (d) Image of LABs-MNs tested on rat skin in vivo. (e) The treated skins were dissected to determine the amount of lactic acid produced in the local skin 2 h following LABs-MNs application. N=2. Data were presented as Mean±SD. Significance was calculated by one-way ANOVA. * p

Transdermal Delivery of Living and Biofunctional Probiotics through

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