Development of a BDDE-crosslinked hyaluronic acid ...

G Model JIEC 3991 No. of Pages 7

Journal of Industrial and Engineering Chemistry xxx (2018) xxx–xxx

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Development of a BDDE-crosslinked hyaluronic acid based microneedles patch as a dermal filler for anti-ageing treatment Jia Nan Zhang, Bo Zhi Chen, Mohammad Ashfaq, Xiao Peng Zhang, Xin Dong Guo* Beijing Laboratory of Biomedical Materials, College of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing, 100029, PR China

A R T I C L E I N F O

Article history: Received 19 March 2018 Received in revised form 27 April 2018 Accepted 9 May 2018 Available online xxx Keywords: Hyaluronic acid Microneedle Cosmetic Anti-ageing Skin

A B S T R A C T

Hyaluronic acid (HA) has many applications in human medicine and cosmetic industry. This study describes the fabrication and evaluation of cross-linked HA (cHA) based microneedles (HA-cHA-MNs) for anti-ageing treatment. The results suggested that the increasing content of cHA gel particles decrease the mechanical properties of MNs. The in-vitro degradation profile and in-vivo epidermal expansion in mice were also carried out. The data suggested that the proportion of cHA particles decreased the rate of MNs degradation. The prepared HA-cHA-MNs have prolonged effectiveness with high swelling retention time or epidermal expansion time in mice up to 6 days or more. © 2018 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.

Introduction Presently, incessant increasing the uses of polymeric constituent in the cosmetics field because of the people’s demands for efficient cosmetics products that might be enhancing the integrity of the skin, without any adverse effects. However, the effectiveness of the commercially available products mainly patches and creams are limited due to skin’s stratum corneum, thereby, relatively limited transportation of the cosmetic products ingredients within the skin. In this context, injectable dermal fillers have attracted considerable attention to combat limited transportation concerns. Significant efforts have been made over the last ten decades to fill and enhance tissue space of the skin by using injectable filler such as autologous fat and paraffin. However, the uses of these fillers are relatively limited because of the serious complications mainly of inflammatory reaction [1–4]. Moreover, polymeric fillers based cosmetic surgery widely used over the last two decade, which diminished the wrinkles and also restores the volume of tissues [5,6]. Several commercially available cosmetics products such as Restylane1, Perlane1, Sub-Q1, Juvéderm1, Surgiderm1, Voluma1 and BellaVita1 [7–10] were widely used for skin treatment. These products used hyaluronic acid (HA) or crosslinked HA (cHA) gel to achieve volume filling, in order to achieve skin moisture and removing wrinkles. However, such injectable or needle-based cosmetic ingredients administration within the skin

* Corresponding author. E-mail address: [email protected] (X.D. Guo).

might be an extremely painful process for the patients, the associated trauma and alleviation of fear, besides the concerns of contamination and disposal [11,12]. On the other hand, hypodermic needles required medical professional. The problem associated with the injectable or needle based cosmetic treatments is being resolved by developing newer mode of injectable delivery system. In this context, microneedles (MNs) based delivery systems has attracted considerable attention [13]. Several cosmetic ingredients such as ascorbic acid, retinyl retinoate, niacinamide, green tea extracts, and n-butylresorcinol, encapsulated polymeric MNs as cosmetic patches are available in the market. Nonetheless, these MNs patches rapidly dissolved, thereby, not effective for longer period [14]. HA is a high-molecular weight, poly-anionic polymer extensively used in the several biomedical applications mainly medicine and cosmetic industry. HA is an unbranched glycosaminoglycan with a disaccharide repeating unit composed of D-glucuronic acid and D-N-acetylglucosamine linked through alternating b-1-4 and b-1-3 glycosidic bonds [15,16]. Naturally, HA is found in skin, extra-cellular matrix (ECM), cartilage, intra-articular joint fluid, vitreous humor and umbilical cord, especially in the skin of the highest content, accounting for half of the total weight of HA [17,18]. It has endogenous, biocompatible, hydrophilic properties, viscoelastic properties, good tolerability and therapeutic, which acts as a good barrier to inflammatory process and a diagnostic marker for many diseases, protects against the effects of free radicals, directs analgesic effect, reduces friction, slows down the evaporation, minimizes risk of adhesions, reduces apoptosis and takes stimulatory action on proteoglycan. Therefore, HA is

https://doi.org/10.1016/j.jiec.2018.05.007 1226-086X/© 2018 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: J.N. Zhang, et al., Development of a BDDE-crosslinked hyaluronic acid based microneedles patch as a dermal filler for anti-ageing treatment, J. Ind. Eng. Chem. (2018), https://doi.org/10.1016/j.jiec.2018.05.007


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J.N. Zhang et al. / Journal of Industrial and Engineering Chemistry xxx (2018) xxx–xxx

commonly used in oncology, orthopaedics, ophthalmology and aesthetic dermatology [19]. The physical and chemical properties of HA are similar with a different source of origin such as animal and bacteria. The only difference is the changes in the chain length or molecular weight. The polymer chain of HA synthesize from bacteria usually shorter than the animal source [19–22]. Due to its unique structure and properties of the HA has gained significant attention and interest over the past few years, especially in the cosmetic industry and aesthetic medicine. HA and its derivative sodium hyaluronate are one of the most common cosmetic ingredients, appears in cosmetic cream formulations and masks, because of the excellent infiltration, moisturizing properties, and it can increase the relative humidity of the skin. Therefore, cosmetic productions contained HA or sodium hyaluronate makes skin appear softer and feel smoother to the touch. However, the consequence of cream formulations or masks is short-term, HA or sodium hyaluronate only covers the stratum corneum and maintaining the moisture of skin, as for the deeper of the skin, it cannot do anything, unless the injection treatment to reconstruct the soft tissue [23,24]. The 1,4-butanediol diglycidyl ether (BDDE) is aware of the most commonly used cross-linking agent in fabrication of cosmetic HA fillers. BDDE is biodegradable, insignificant toxicity than other ether-bond crosslinking agents, thereby, safer for biomedical application mainly cosmetic products [25]. The present study describes the fabrication of cosmetic BDDEcrosslinked HA gel with HA molecules based MN (HA-cHA-MNs) patch for cosmetic application mainly ageing treatments. The prepared HA-cHA-MNs was applied to various parameters namely morphological analysis, mechanical strength measurement, skin penetration ability, in-vitro biodegradation study and in-vivo epidermal expansion/biodegradation assay, and fluorescence microscopy. The novelty of the present study as follows: the formulation of BDDE cross linked with the HA enhances the mechanical strength, in-situ swelling ability of the MNs within the skin and decrease biodegradability of HA. The in-situ swelling ability and slow biodegradability of the prepared HA-cHA-MNs might be beneficial for prolonged effectiveness of the dermal filler. To the best of our knowledge, there have been no HA based MN patches with BDDE-crosslinked HA gel particles with HA molecules for anti-wrinkle treatment. This is the first study showing the use of the BDDE-crosslinked HA gel particles based MNs with efficient applicability towards the anti-wrinkle treatments. Therefore, the study will have a significant impact on MNs application for cosmetic industry and anti-ageing research. Experimental section Fabrication of cHA Materials HA (MW = 447.37 Da) was purchased from Toronto Research Chemicals Inc. (Toronto, Canada), BDDE (1,4-butanediol diglycidyl ether, MW = 202.25 Da) was purchased from Adamas Reagent Co, Ltd. (Basel, Switzerland), Sulforhodamine B (fluorescein; MW = 558.666 Da), Phosphate buffered saline (PBS) and Deuterium oxide (D2O, D > 99.9%) were purchased from Sigma-Aldrich (St. Louis, MO, USA), Hyaluronidase (300 units/mg) and Alcohol (concentration: 95%) were purchased from Aladdin Industrial Corporation (Shanghai, China), Ehrlich’s reagent was purchased from BioTechnology Co, Ltd (Shanghai, China), Acetic acid and sodium carbonate anhydrous (Na2CO3) were obtained from Sinopharm Chemical Reagent Co, Ltd (Bejing, China) and Polydimethylsiloxane (PDMS; Sylgard 184) was obtained from Dow Corning (Midland, MI, USA). Female Balb/c mice were obtained from the Institute of Laboratory Animal Sciences (ILAS). Aqueous solutions were carried

out using ultrapure water from a reverse osmosis membrane system unless otherwise stated. Fabrication of cHA hydrogel particles Cross-linking HA (cHA) reagent solution was prepared by mixing 200 mL of 1,4-butanediol diglycidyl ether (BDDE) into 9.8 mL of 0.25 M NaOH, (pH 13). Approximately 1.0 g of HA powder was added to the cHA reagent solution and mixed thoroughly at 40 C for 2 h [26]. After the cross-linking, the mixed solution contains non-reacted NaOH, BDDE and HA fragments. HA can be purified with 95% ethanol. The prepared hydrogel was ground, squeezed out and screened with a 300 mesh sieve (GB/T6003.12012) to get particles having a diameter of less than 50 mm. Fig. 1 shows the chemical reaction between cHA reagent and BDDE cross-linker under alkaline condition. The opening of BDDE rings during reaction reacts with the –OH group of HA to produce crosslinked network (hydrogel). Nuclear magnetic resonance spectroscopy (NMR) of cHA For NMR spectroscopy the prepared cHA and HA were incubated with hyaluronidase for complete digestion. Each fraction of digestion was centrifuged (Centrifuge 5702 RH, Eppendorf, Hamburg, Germany) at 4000 rpm for 5 min. Next, the supernatant was collected and vacuum dried. 1H NMR analysis of each fraction (dissolved in 0.55 mL D2O) was done using a Bruker 600 MHz (Zurich, Switzerland) instrument [27]. The spectra was acquired by MestReC software. Fabrication of HA-cHA-MNs Preparation of PDMS molds The PDMS micromolds were fabricated using a laser-based micromolding technique as discuss in our previous study [28]. Briefly, the PDMS was mixed in a 10:1 v/v ratio of prepolymer to curing agent and degassed under an 800 Mb vacuum for 30 min to eliminate entrap air bubbles, and then cured into a customized mold with smooth surfaces at 60 C for 5 h for the preparation of PDMS sheet. Next, the fabrication of micro-cavities on PDMS sheet by using laser engraving machine (Universal laser system, Inc., Scottsdale, Arizona, USA) to prepare PDMS mold. In this study, the micro-cavities on the mold were patterned into 5 5 and 10 10 with depth of 650 mm. Fabrication of HA-cHA-MNs The different weight ratio of the HA and cHA (HA-cHA) (1:1), and (5:1) were used for the fabrication of HA-cHA-MNs, where (1:0) and (0:1) indicate only HA and cHA based MNs, respectively. The mixtures of different weight ratio of the polymer were dissolved in ultrapure water and a 20% (w/v) viscous polymer solution was stirred at 360 rpm at room temperature under a magnetic stirrer (SCILOGEX, LLC, USA) for the production of homogenous suspension. First, the mold cavities were filled with the homogenous solution under vacuum pressure and cleaned by the tape. The viscous solution was applied to the mold, and the residual solution remaining on the edge of the mold was peeled off by using a blade. After overnight curing at room temperature, the cured MNs patch was successfully prepared by separation from the mold using an adhesive PMMA plate (Fig. 2). Mechanical property test Axial strength of HA-cHA-MNs The mechanical property tests of different ratio of HA-cHA-MNs were carried out similar to our previous study [29], by using a motorized force measurement test stand (ESM301, Mark – 10, Force Gauge Model, USA) to measure the axial strength of one MNs,




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Fig. 1. The chemical reaction between cHA reagent and BDDE crosslinker.

Fig. 2. Schematic illustrations of fabrication process of HA-cHA-MNs and its application.

in order to indicate mechanical properties of the three kind of MNs. Briefly, MNs was compressed between two metallic parallel plates in a vertical direction. A MN (650 mm height) was placed on the horizontal platform, and then we applied vertical stress of dynamometer probe at a speed of 0.5 mm/min. The probe contacts the topmost point of the MNs, the testing machine displayed compression force, and analyzed the data using Mask – 10 software. Insertion capability of HA-cHA-MNs In-vitro skin test was performed to indicate the insertion ability, in order to compare the mechanical property of different ratios of MNs prepared in this study. The MNs patches were inserted in thick porcine cadaver skin. The hairs of skin were carefully removed by using disposable shaver. The porcine cadaver skin was procured from a local slaughter house (Beijing, China) and separated into 10 cm 10 cm square without subcutaneous tissue samples, used as a human skin model. A, 5 5 HA-cHA-MNs patches of different ratios of HA and cHA were inserted in porcine cadaver skin for 5 s, and then sulfonylrhodamine B (model drug) solution was coated on the insertion site of skin for 2 min after the removal of MNs. Next, the remaining drug solution was clean which was not adsorbed in the cavities of skin. After cleaning of unabsorbed drug solution, insertion ability of MNs in skin samples was observed under the fluorescence microscope (SZX7, Olympus Optical co. Ltd, Tokyo, Japan).

Effect duration of HA-cHA-MNs In vitro MNs degradation rate In-vitro MNs degradation rate was measured using a colorimetric method. The measurement is based on the degree of degradation of the N-acetyl glucosamine (NAG) that does not change during the reaction time. The different prepared matrix material such as HA-cHA (1:0), (0:1), (1:1), and (5:1) were used for the degradation analysis. Approximately 500 mL of 300 units/mL hyaluronidase mixed in 3 mL of PBS solution and incubate for different time intervals (1–5 days) at 50 C. The samples of each material were kept for undetermined time for complete degradation served as control. After incubation time, each sample was heated in boiling water for 10 min to stop the enzymatic reaction and then centrifuge at 4000 rpm for 5 min. After centrifugation collect the supernatant and mixed with 5 mL of PBS. Approximately 100 mL of 0.25 M sodium carbonate was mixed with 1 mL of diluted supernatant solution and incubated in water bath at 100 C for 1 min. After incubation period, solution was titrated using the mixed solution of glacial acetic acid and Ehrlich’s reagent mixture and left until violet colour was produced. The absorbance was recorded at 585 nm using spectrophotometer. All experiment was repeated to ensure the repeatability and data represented as mean with a 95% confidence interval. The in-vitro degradation rate for each sample was expressed as NAG, CNAG represented the complete degradation and PNAG is the



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quantity of degradation for each sample. The equation of in vitro degradation rate of three MNs was: Degradation NAG = PNAG/CNAG 100% In vivo MNs effect in mice Female Balb/c mice weighing 20 2 g were used for the in-vivo experiments. The animals were acclimatized to the laboratory conditions for approximately two weeks before the any experiment. Institutional animal care and committee of Beijing University of Chemical Technology approved the all experiments throughout this study were conducted according to the guidelines of the Laboratory Animal Centre of Beijing University of Chemical Technology, Beijing, China. The mice were anesthetized by using 2.5% isoflurane. Next, the hair on the back of the mouse was removed with an electric shaving machine and the back skin was kept clean with a depilatory cream. The prepared different ratios of HA-cHA-MNs were manually inserted the different parts in mouse, and removed after 5 min. The degree of swelling subcutaneously and duration were observed by digital microscope (AD7013MZT, Dino-lite, Taiwan, China). In vivo inserted depth in the mice skin We observed whether the microneedles penetrated subcutaneously successfully by observing the penetration depth of the MNs in the skin of the mice, and thereby determined whether MNs was the reason for epidermal expansion. The experimental material we used was HA MNs loaded with sulforhodamine B. The MNs were applied to the epidermis of mice according to the same procedure as in the appeal test. The mice were then sacrificed. The epidermis was taken and longitudinal sections of the skin were cut along the needle holes. Microscopic photographs of the bright and fluorescence fields of the corresponding sections were taken with a fluorescence microscope. Results and discussion Modification degree of cHA

the basis of NMR spectra, approximately 8% degree of modification in cHA was observed. The degree of modification is significantly higher or comparable with other commercially available products, which was vary between 1% and 8% for the HA fillers [30]. Therefore, the prepared cHA gel was lies within the standard limit of modification degree of the dermal fillers. Characterization of MNs In the present work, we fabricated HA, cHA and mixture of HA and cHA based MNs for anti-ageing treatment. HA is commonly used in the skin treatment or cosmetic products due to its unique properties such as non-toxic in term of genetic, reproductive, and developmental, and does not show any immune response, as discuss earlier in the text [31]. Therefore, HA and HA based composite is safer for end application. On the other hand, HA and different ratio of HA-cHA-MNs were used for the fabrication of different MNs due its sufficient mechanical strength, resistance to deformation and rapid dissolution in skin, thereby, easily penetrate within the skin. The vacuum fabricating process was used in the present study. Using this fabrication process, we successfully fabricated HA and different ratio of HA-cHA-MNs arrays consisted of 25 (5 5) and 100 (10 10) MNs with the height of 650 mm, base diameter of 250 mm, the diameter of tip of needle is 10–15 mm and centre to centre spacing of 700 mm and 500 mm. All of the above types of MNs have the same appearance characteristics. Fig. 4, shows the bright-field microscopic image of HA-cHA-MNs patch that contains 100 (10 10) MNs. The fabrication process is facile and do not require any complicated instruments, thereby, suitable for large-scale production. Mechanical property of polymer MNs The sufficient mechanical strength of the MNs is important characteristic for its successful application, thereby, reliable insertion within the skin without breakage or bending of tip during application. The prepared different ratio of HA-cHA was used for the mechanical strength of the MNs, using both axial mechanics and the ability of insertion within the skin to evaluate the feasibility of its usage in as a substitute for cosmetic fillers.

Fig. 3 shows 1H NMR spectra of before and after cross-linking of HA using BDDE. The N-acetyl glucosamine peak at 1.945 ppm was observed in both HA and cHA samples. The two new emerging peaks at 1.8 and 1.558 ppm were observed in BDDE cross linked sample cHA attributed the unsaturated bond in the unsaturated disaccharide (DDiHA) [27], and –CH2 of the BDDE molecule, respectively. The presence of both peaks in cHA sample indicated the modification in HA molecules due to BDDE cross-linking. The NMR result indicated a characteristic peak at 1.558 ppm in the cHA degradation fraction spectra which was absent in HA spectra. On

Measurement of axial strength The prepared different ratios of HA-cHA were measured the mechanical properties of the MNs by testing the axial strength of a single MN. All MNs have similar geometry and aspect ratio to ascertain the mechanical properties of different matrix materials of MN. Fig. 5 shows the mechanical behaviour (force displacement

Fig. 3. 1H NMR spectra of HA and cHA degradation fraction.

Fig. 4. The bright-field micrographs of HA-cHA-MNs patch.




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Skin insertion capability of MNs The prepared HA and different ratios of HA-cHA based MNs were used for ascertaining the skin insertion capability and sulfonylrhodamine B drug was used as a model drug for corroborated the penetration ability with the skin. Fig. 6 shows the bright field and fluorescence microscopic images after insertion of different polymers based MNs. The red spot in bright images and fluorescence intensity in HA and HA-cHA (5:1) is approximately same, whereas HA-cHA (1:1) MN sample shows the marginally lower. These data suggested that all variation of MNs have sufficient insertion ability within the skin. The data suggested the similar trends as observed in the measurement of axial strength, discuss earlier in the text. Biodegradation of polymeric matrix of MNs

Fig. 5. Mechanical behaviour of different ratio of HA-cHA-MNs.

curve) of different matrix materials based MN. The data clearly indicated that the force-displacement curve of (1:0) based MN had higher bending strength, thereby, exceptional mechanical strength among all of them. Whereas, the force displacement curve decrease with decrement of HA content or increment in cHA particles in the polymeric matrix of MN due to the heterogeneous structure. The different structure of HA and cHA in HA-cHA-MNs weaken the mechanical stability of the MN. The research study also suggested that the mechanical properties of MNs which had heterogeneous structure decrease with the increasing the proportion of the mixed particles, however, only few degree of mechanical properties was sufficient to penetrate the within the skin [32]. Although, the mechanical properties of HA-cHA (1:1) had the lowest mechanical strength than others, but still have some mechanical strength. Therefore, the prepared HA and different ratio of HA-cHA based MNs have sufficient insertion ability within the skin. We revisit this aspect to ensure the real time applicability in the subsequent section.

The stability or degradation behaviour of the dermal filler is essential factors in anti-ageing application. The longer stability or slow degradation of filler materials indicated more effectiveness of the filler materials. Therefore, the in-vitro and in-vivo degradation test of the prepared different ratios of HA-cHA-MNs were carry out to ascertain the effectiveness of the matrix materials of MNs. In vitro MNs degradation rate Fig. 7 shows the degradation percentage of the different ratios of HA-cHA-MNs with time. The highest degradation rate was observed in (1:0), whereas, (0:1) have the lowest degradation rate. The degradation rate decrease with increasing the cHA content in matrix materials of MNs. As observed from the figure, approximately 65% degradation was observed in HA based MNs within a first day of experiment, and the complete degradation was observed within three days of experiment. On the other hand, approximately 35% degradation rate was observed in cHA based MNs with respect to HA and almost 50% polymeric matrix remaining after five days of test. The data suggested that the ratio of cHA increased in HA, decrease the degradation rate. Therefore, different ratios of cHA based MNs have potential ability for dermal

Fig. 6. In-vitro skin penetration ability of the different ratio of HA-cHA based MNs on porcine cadaver skin using sulfonylrhodamine B model drug. (a). bright-field and (b) fluorescence microscopic images.



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filler applications. We revisit this aspect in in-vivo test analysis of MNs in a subsequent section.

Fig. 7. Degradation NAG-time curve of different ratio of HA-cHA-MNs. The data represent an average value (n = 3) standard error (SE).

In-vivo MNs effect in mice Fig. 8 showed the in-vivo effect of MNs on the skin of mice using different ratio of HA-cHA. The images clearly showed that epidermal expansion in all matrix materials based MNs. Fig. 8(a1–c1) showed the images at time of insertion that indicated the all polymeric matrix based MNs have sufficient insertion ability, which also confirmed from mechanical strength and invitro skin penetration ability. Fig. 8(a2–c2) images showed that maximum epidermal expansion was observed in HA. The relative epidermal expansion in of the different polymers based MNs array within a first hour of experiment is as follows: HA > HA-cHA (5:1) > HA-cHA (1:1). However, the epidermal expansion using HA based MN was disappeared after 2 days of experiments indicated the degradation of HA molecules (Fig. 8a3). The epidermal expansion was eliminated at 4 days of experiments in HA-cHA (5:1) based MNs (Fig. 8b4), whereas in HA-cHA (1:1) sample

Fig. 8. Photographic images of In-vivo epidermal expansion analysis using different polymeric matrix based MNs (a) HA. (b) HA-cHA (5:1) (c) HA-cHA (1_1) and different days (a1–c1). 0 h, (a2–c2). 1 h, (a3–c3) 2 days, (a4–c4). 4 days, and (a5–c5). 6 days of experiments.

Fig. 9. The corresponding section of mice skin after removal of HA MNs using sulfonylrhodamine B model drug.




epidermal expansion was stable up to four days and eliminated at six days of experiment. The epidermal expansion was smaller on increasing cHA content in matrix materials of MNs. The expansion of the polymeric matrix is mainly depends on the water adsorption ability that decrease with the cross-linking of cHA. The in-vivo test analysis data was consistent with in-vitro experiments. The higher stability or lower degradation rate of polymeric matrix shows the potential ability of matrix materials for dermal filler or anti-ageing application. In vivo inserted depth in the mice skin Fig. 9 shows the micrograph of the longitudinal section of the needle holes after the MNs penetrated into the mice skin. It can be seen from the figure that the inserted depth of the MNs is about 350 mm, and the epidermal layer of the mice has been penetrated. The penetration of the needle is effective and enables the microneedle to function subcutaneously. Therefore, the reason for the epidermal expansion was the function of the MNs. Conclusion The different ratio of HA-cHA based MNs such as (1:0), (0:1), (5:1) and, (1:1) were successfully fabricated for anti-ageing treatment. The mechanical strength and skin penetration ability suggested that the fabricated MNs possess suitable mechanical strength, thereby, efficiently penetrate in the skin. The 1H NMR spectra also confirmed the degree of modification due to crosslinking of HA with BDDE. The in-vitro and in-vivo degradation test assay confirms stability and effectiveness of the polymeric matrix over a prolonged period. The data suggested that different ratio of polymeric matrix HA-cHA-MNs might be suitable for dermal filler applications. In general, the prepared MNs have a potential that might be efficiently used as derma filler in cosmetic or anti-ageing treatment that might be replaced aesthetic surgery.

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This work was financially supported by the National Natural Science Foundation of China (51673019) and the long-term subsidy
