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In situ-formed cryomicroneedles for intradermal cell delivery – NPG Asia Materials

Time Stamp: February 22, 2024 7:00 PM
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Fabrication and characterization of porous MN scaffolds

The porous scaffolds used to create S-cryoMNs were made by lyophilizing hydrogels in the MN template. As a proof-of-concept, we used methacrylated hyaluronic acid (MeHA) to demonstrate this principle. Hyaluronic acid (HA) with different molecular weights (48 kDa and 300 kDa) was first modified with methacrylic anhydride to obtain photocrosslinkable 48-MeHA and 300-MeHA. The degrees of substitution, evaluated by proton nuclear magnetic resonance (¹H NMR), were 93.6% and 70.3% for 48-MeHA and 300-MeHA, respectively (Fig. S1). Later, an aqueous solution of MeHA was used to fill the negative polydimethylsiloxane (PDMS) MN mold, which was photocrosslinked before freeze-drying (Fig. 2A). Here, the ice was the porogen that introduced the micropores in the MN structure^7,8,9. The morphology of the derived MN scaffold was similar to that of the original MN master template. While the stainless steel master MN template had a height of 1200 μm and a base width of 300 μm (Fig. S2), the height and base width were 700 μm and 270 μm, respectively, for the porous MNs made of 48-MeHA, and 620 μm and 320 μm, respectively, for the MNs made of 300-MeHA. Changes in the MN scaffold dimensions, which are due to the shrinkage of PDMS and the polymeric matrix, have been observed in many previous studies^10,11,12.

The porous structure of the MNs was tunable by adjusting the MeHA concentration and photocrosslinking time. We examined the changes in morphology of the MN scaffolds made with different MeHA concentrations (2, 4, 5, and 6 wt% 300-MeHA and 2, 4, 6, and 8 wt% 48-MeHA) with the same crosslinking time (5 min). As shown in Fig. 2B, C, 300-MeHA MNs provided a porous structure when the concentration of the polymer was 4%. Similar results were obtained for 48-MeHA MNs at 4% and 6% wt% 48-MeHA. Lower concentrations of 48-MeHA and 300-MeHA did not provide a stable MN structure, while higher concentrations significantly decreased the porosity^6,13. The crosslinking time is another important parameter during fabrication. With the optimized MeHA concentrations (4% for 300-MeHA and 6% for 48-MeHA), we examined UV crosslinking times of 3 min (CL3), 5 min (CL5), 10 min (CL10), and 20 min (CL20) (Fig. 2D, E). In general, the longer the UV exposure time was, the lower the porosity was¹⁴. Specifically, when the crosslinking time was 10 min or longer, we obtained a shell structure instead of a porous structure. This shell structure is not preferrable for loading cells.

Therefore, CL3 and CL5 of 48-MeHA (CL3-48-MeHA and CL5-48-MeHA) and CL3 of 300-MeHA (CL3-300-MeHA) were identified as the optimal formulations due to their intact MN structure and observable porosity. We further analyzed the porosity and dimensions of the samples via the SEM images using ImageJ. The CL3-48-MeHA and CL5-48-MeHA MNs had average MN heights of 697.1 ± 21.0 μm and 682.9 ± 10.7 μm, respectively. The CL3-300-MeHA MNs were slightly shorter at 616.4 ± 32.8 μm. The average pore sizes for CL3-48-MeHA, CL5-48-MeHA, and CL3-300-MeHA were 81.0 ± 36.8 μm, 56.6 ± 22.8 μm, and 54.2 ± 23.9 μm, respectively. These pore sizes match well with that of mammalian cells, which are usually 10–30 μm in suspension. Of these three hits, CL3-300-MeHA was used as a representative for the following studies.

While the rest of the work was done with the MeHA porous MN scaffold, it should be noted that other hydrogel-derived scaffolds can also be used. As shown in Fig. S3, porous MN scaffolds may be prepared from other hydrogel formulations, including but not limited to cryogelation of polyvinyl alcohol (PVA), cryogelation of gelatin (gelatin), EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide)-crosslinked gelatin (EDC/NHS-Gel), 1,4-butanediol diglycidyl ether crosslinked hyaluronic acid (BDDE-HA), photocrosslinked gelatin methacryloyl (GelMA), and photocrosslinked polyethylene glycol diacrylate (PEGDA).

Cell loading and release from S-cryoMNs

S-cryoMNs are made by dipping the porous MN scaffold in a cell suspension and freezing. Like in the first generation of cryoMNs, the cryogenic medium is the key factor for maintaining cell viability and function in the device. Dimethyl sulfoxide (DMSO) is the most popular cryoprotective agent because it binds water molecules to prevent ice crystallization and cell damage^15,16. However, the commonly used concentration (10%, v/v) can be toxic to cells and induce cell membrane damage and gene expression alterations¹⁷. During the intradermal delivery of therapeutic cells, a high dose of DMSO might cause unwanted side effects on both the therapeutic cells and surrounding skin cells.

Macromolecular cryoprotectants, in which hydrophilic macromolecules bind with water and increase solution viscosity to inhibit extracellular ice growth, have been reported to be a partial substitute for DMSO to reduce toxicity¹⁸. Hydrogel-forming polymers are macromolecules characterized by good biocompatibility and good affinity for water molecules. Some of these materials, such as polyethylene glycol (PEG) and hydroxyethyl starch (HES), have been applied in the cryopreservation of red blood cells^19,20, stem cells^21,22,23, and other mammalian cells^24,25,26. Therefore, hydrogel polymers are considered potential candidates for replacing some of the DMSO to develop cryoprotective media with low toxicity.

We screened eight hydrogel-forming polymers that have been used to cryopreserve cells: PEG, HES, methylcellulose (MC), sodium carboxymethyl cellulose (CMC), HA, chitosan, gelatin, and alginate. These polymers were mixed with low doses of DMSO (1% and 2%) as cryogenic media for the cryopreservation of human dermal fibroblasts. Among the eight candidates, cryogenic media containing PEG, HES, MC, and CMC ensured cell survival (Fig. S4). Later, the concentrations of PEG, HES, MC and CMC were further evaluated from 0, 0.25, 0.5, 1.0 to 2.5 wt% (Fig. S5). When the concentration of DMSO was lower than 1%, the cryogenic solution did not sufficiently protect the cells during cryopreservation. With 2% DMSO, all cryogenic solutions of the four polymers provided similar or even greater cell viability than 10% DMSO. We further confirmed the preservation ability of these four best formulations (2.5 wt% PEG, 2.5 wt% MC, 2.5 wt% CMC, and 0.25 wt% HES with 2.0% DMSO) on three other cell types, namely, human keratinocytes (HaCaT cells), a human malignant melanoma cell line (A375), and human bone marrow-derived MSCs. As shown in Fig. 3A, the fibroblasts, A375 cells, and HaCaT cells maintained comparable, if not greater, viabilities in these cryogenic formulations compared to those in classic cryogenic media supplemented with 10% DMSO (except 2% DMSO + 0.25% HES for HaCaT cells). However, no advantage was observed for hMSCs except for 2% DMSO + 2.5% CMC. However, compared with media supplemented with 10% DMSO, cryogenic solutions containing PEG, HES, MC, or CMC did not induce any significant toxicity (Fig. S6). Finally, the formulation containing 2.5% CMC and 2% DMSO was ultimately selected as the representative cryogenic solution for subsequent cell loading.

**Fig. 3: Cell loading and release from S-cryoMNs.**

The representative porous MN scaffold (CL3-300-MeHA) was immersed in cryogenic medium containing cells (A375 cells as an example) for 1 min. The loading of cells into the porous MN scaffold was driven by capillary force during wetting. After the scaffold was prewetted, the cells could not reach the matrix (Fig. S7A). The polymeric component (CMC) increased the viscosity of the cryogenic solution and thus reduced the climbing viscosity of the solution inside the scaffold²⁷, which was beneficial for uniformly distributing cells in the porous scaffold (Fig. S7B).

Later, the cell-loaded S-cryoMN patches were frozen in a cryopreservation box. After overnight freezing, the patches were stored at −80 °C for the short-term or in liquid nitrogen for longer storage (>7 days). When the frozen S-cryoMNs were placed at room temperature (24 °C), they melted and completely thawed within 2 min (Figs. 3B and S8). The encapsulated A375 cells had an average viability of 76.3 ± 7.7% (Fig. 3C). Cells labeled with green dye were observed inside the tips of the S-cryoMNs (Fig. 3D), and the number of loaded cells inside the S-cryoMNs was tuned according to the initial cell density in the cell suspension. The release of cells from S-cryoMNs was tested on an agarose skin phantom. When the cell concentration was increased from 2.5 × 10⁶ to 5.0 × 10⁶, 7.5 × 10⁶, and 1.0 × 10⁷ cells/ml in cryogenic medium, the number of cells released from each needle increased accordingly from ~10 cells/needle to 240 cells/needle (Fig. 3E, F). The loaded cells were released from the S-cryoMNs upon melting and degradation (Fig. S9A). The degradation behavior of the porous MN scaffold could be efficiently tuned by adjusting the second crosslinking degree to achieve a desirable release profile (Fig. S9A, B). In the following studies, a second crosslinking cycle of 8 min was adopted to achieve relatively rapid release within 8 h.

As shown in Fig. 3G, H, the S-cryoMNs easily penetrated the agarose gel and delivered the stained A375 cells into the agarose gel at a depth of ~200 μm. The mechanical strength of the S-cryoMNs was tested via a compressive mechanical test in which the S-cryoMNs could bear 0.058 N of force with a single needle, which indicated that the strength of the S-cryoMNs was sufficient for skin penetration²⁸ (Fig. S10). Skin penetration was also validated on ex vivo porcine skin, where the S-cryoMNs were easily inserted into the skin, and an approximate penetration depth of 450 µm was determined from the histological images (Fig. S11). Note that the agarose gel is much more elastic than porcine skin is; thus, the S-cryoMNs had less penetration into the gel phantom.

Using S-cryoMNs for the delivery of allogeneic and autologous cells

In cell therapies, the cells to be delivered might originate from the patient (autologous cells) or a donor (allogeneic cells). These therapeutic cells are collected, expanded, and engineered ex vivo before administration to the patient. The S-cryoMNs utilize a dip-loading procedure that allows their on-site preparation for subsequent cell delivery as well as long-term storage for later application (Fig. 4A). Here, we used hMSCs and melanocytes as model cells to demonstrate the potential applications of S-cryoMNs for delivering both allogenic cells and autologous cells.

**Fig. 4: In vitro biological functions of cells delivered using S-cryoMNs.**

MSCs are promising therapeutic cells because of their immunosuppressive and tissue repair properties²⁹. Autologous MSCs can suffer from insufficient quantities, decreased biological activity (elderly individuals as donors), or impaired functions (donors with systemic diseases). Using allogeneic MSCs from young healthy donors is a reasonable approach to resolving these issues. Here, we loaded 1 × 10⁵ hMSCs into S-cryoMNs and examined their potential application in wound healing through scratch and tube formation assays³⁰. A scratch assay was used to examine the migration capability of dermal fibroblasts in response to hMSCs released from the S-cryoMNs. We first examined the migration and proliferation of fibroblasts into the wounded area at 24 and 48 h after treatment under four conditions: low serum concentration (1% FBS instead of 5% FBS in the other groups), positive control (20 ng/ml TGF-β1), blank S-cryoMNs, and hMSC-loaded S-cryoMNs. As shown in Figs. 4B and S12, treatment with S-cryoMNs significantly promoted the migration of fibroblasts. Closure was 50.8 ± 6.1% at 24 h and 86.5 ± 5.0% at 48 h in the cell-loaded S-cryoMN group, while closure was 25.5 ± 11.4% at 24 h and 59.1 ± 7.6% at 48 h for the group treated with blank S-cryoMNs. This result is comparable to that of the positive control. We also carried out a tube formation assay under four different conditions: low serum concentration (1.5% FBS instead of 5% FBS in the other groups), a positive control (40 ng/ml VEGF), blank S-cryoMNs, and hMSC-loaded S-cryoMNs. Eight hours after treatment, a tube-like network was clearly observed in the cell-loaded S-cryoMN group (Fig. S13). In the group treated with hMSC-loaded S-cryoMNs, there was a 2.3-fold increase in the total tube length compared to that of the group treated with blank S-cryoMNs (Fig. 4C). Treatment with hMSC-loaded S-cryoMNs increased the numbers of tubes and branching nodes by 2.7 times and 2.0 times, respectively, compared to those in the blank S-cryoMN group.

Vitiligo is a skin depigmentation disorder caused by the autoimmune destruction of melanocytes, which results in a loss of melanin expression^31,32. Autologous melanocyte transplantation is a surgical strategy for regenerating melanocytes and repairing vitiliginous areas^33,34. As melanocytes typically reside in the basal epidermis and dermal junctions³⁵, recipient sites are commonly exposed to the papillary dermis by invasive abrasion methods (laser abrasion³⁶, dermabrasion^37,38, and the suction blister method³⁹) to ensure the survival of melanocytes after transplantation. Here, we used B16-loaded and melanocyte (Mela)-loaded S-cryoMNs to restore melanin expression in model mice. The B16-loaded S-cryoMNs (characterization details are shown in Fig. S14) were applied to the backs of the mice via a thumb press to intradermally deliver B16 cells, and the results from this method were compared with those from the dermabrasion technique (Fig. 5A). As shown in Fig. 5B, C, 1 day after cell transplantation via B16-loaded S-cryoMNs, the delivered cells could be visualized at the base of the epidermis and dermis, ranging from ~50 μm to 200 μm. On day 3, the melanin production by the delivered B16 cells was comparable to that of the dermabrasion group.

**Fig. 5: In vivo delivery of cells that secrete melanin using B16-loaded and Mela-loaded S-cryoMNs.**

We also applied melanocyte (Mela)-loaded S-cryoMNs (characterization details are shown in Fig. S14A, B) for intradermal transplantation of melanocytes. On day 0, each mouse received four treatments on four different dorsal sites: no treatment, Mela-loaded S-cryoMNs, blank S-cryoMNs, and dermabrasion transplantation (Fig. 5A). In the dermabrasion treatment, the epidermis was mechanically removed to expose the superficial dermis to obtain a melanocyte suspension with a density of ~250 cells/mm², which was consistent with that of Mela-loaded S-cryoMNs (~220 cells/mm²)^40,41. The Mela-loaded S-cryoMNs were pressed onto the mouse skin and not removed until the patches had melted and softened. On day 1 after treatment, the melanocytes released from the S-cryoMNs were visualized in the mouse skin from the epidermis to the dermis (depth up to ~300 μm), and these cells were found mostly in the basal layer of the epidermis and around the hair follicles (indicated by the red arrows in Fig. 5D). Three days after S-cryoMN transplantation, the delivered melanocytes remained viable and produced melanin. Moreover, there was no significant decrease in the number of cells according to semiquantitative analysis of the histological images (Fig. 5E). The efficacy of the Mela-loaded S-cryoMNs was similar to that of the dermabrasion technique in terms of intradermal transplantation of melanocytes. In contrast, the dermabrasion method was markedly more invasive, as the epidermis was destroyed on day 1 and a scab had formed on day 3, as shown in Fig. 5D.

OVA-DC S-cryoMNs for cancer vaccination in vivo

DCs are potent antigen-presenting cells (APCs) that capture and present antigens to T lymphocytes for long-lasting and specific immune memory. Additionally, autologous DCs have been applied for the development of vaccines in the clinic. We developed DC-loaded S-cryoMNs and tested their potential for use in vaccination in a mouse model. DCs were pulsed with ovalbumin (OVA) as a model antigen⁴, after which the surface markers of mature and active DCs were examined (CD11c, CD86, and MHCII). Lipopolysaccharide (LPS)-pulsed DCs served as the positive control (Fig. S15A). Specifically, OVA stimulation generated 63.5 ± 6.9% CD11c⁺CD86⁺ DCs and 68.8 ± 9.1% CD11c⁺MHCII⁺ DCs, which was comparable to the LPS-pulsed DCs (72.4 ± 12.0% CD11c⁺CD86⁺ DCs and 74.6 ± 8.2% CD11c⁺MHCII⁺ DCs). After being loaded into S-cryoMNs (OVA-DC S-cryoMNs), the OVA-DCs maintained high viability after short-term (83.1% after 1 week at −80 °C) and long-term storage (77.2% after 1 month of storage in liquid nitrogen and 63.4% after 3 months of storage in liquid nitrogen) (Fig. S15B, C).

We optimized the dosage and application frequency of OVA-DC S-cryoMN vaccination. Healthy mice were subcutaneously (s.c.) vaccinated twice a week with different dosages of OVA-DCs (0, 1, 2, or 8 × 10⁵). The levels of OVA-specific antibodies in the blood significantly increased in the third week and became saturated after eight treatments (Fig. S16). The 2 × 10⁵ OVA-DC dosage produced the best antigen-specific immune response. Therefore, we used S-cryoMNs with 2 × 10⁵ OVA-DCs (two patches per dose with each patch containing 1 × 10⁵ OVA-DCs) to vaccinate the mice over 4 weeks for a total of 8 treatments (Fig. 6A, B). OVA-DC S-cryoMNs penetrated mouse skin up to ~200 µm in the dermal area, as shown in Fig. 6C. After one treatment with OVA-DC S-cryoMNs, prelabeled OVA-DCs were observed in the draining lymph nodes (Fig. S17A, B). On day 28, the mice were inoculated with B16-OVA melanoma cells. The tumors were measured every 2 days and excised on day 24 post inoculation. As shown in Figs. 6D, E and S18, tumor growth was much slower and the tumors were much smaller in mice inoculated with OVA-DC S-cryoMNs (average size of 186.1 ± 144.3 mm³ and weight of 135.2 ± 114.7 mg) compared with mice treated with blank S-cryoMNs (average size of 905.8 ± 445.1 mm³ and weight of 1037.1 ± 662.2 mg).

**Fig. 6: In vivo vaccination using ovalbumin-pulsed dendritic cell-loaded S-cryoMNs (OVA-DC S-cryoMNs).**

The vaccinated mice had a greater percentage of CD11c⁺CD86⁺ DCs (2.16 ± 0.23% vs. 1.22 ± 0.24% in the blank S-cryoMNs group) and more CD11c⁺MHCII⁺ DCs (1.04 ± 0.32% vs. 0.72 ± 0.26% of the blank S-cryoMNs group) in the excised draining lymph nodes (Figs. 6F and S19). After splenocytes were collected from vaccinated mice and restimulated with OVA, OVA-DC S-cryoMNs-vaccinated mice exhibited a significantly greater proliferation rate (~1.3-fold greater than that of the blank S-cryoMNs group; Fig. 6G) and greater secretion of IFN-γ (736.4 ± 145.1 pg/ml vs. 202.1 ± 33.0 pg/ml in the blank S-cryoMNs group; Fig. 6H). As shown in Fig. 6I, immunization with OVA-DC S-cryoMNs induced significantly greater OVA-specific toxicity to splenic T lymphocytes than vaccination with blank S-cryoMNs. The above results suggested that vaccination with OVA-DC S-cryoMNs could induce potent antigen-specific immune responses with strong antitumorigenic effects.

The S-cryoMNs were safe throughout the entire experimental period. All of the thumb-pressed S-cryoMNs left a clear micropattern on the mouse skin, which visually disappeared within 1 h (Fig. S20A); there was no obvious skin damage. After 8 applications of S-cryoMNs over 1 month, we excised the skin and performed H&E staining and did not observe any increase in the infiltration of inflammatory cells in the skin tissue (Fig. S20B). Such frequent administration of S-cryoMNs induced neither weight loss (Fig. S20C) nor pathological damage to major organs (Fig. S20D).

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Source: https://www.nature.com/articles/s41427-024-00531-1

In situ-formed cryomicroneedles for intradermal cell delivery – NPG Asia Materials

Fabrication and characterization of porous MN scaffolds

Cell loading and release from S-cryoMNs

Using S-cryoMNs for the delivery of allogeneic and autologous cells

OVA-DC S-cryoMNs for cancer vaccination in vivo

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