A systematic review of carbohydrate-based microneedles: current status and future prospects

Microneedles (MNs) are minimally invasive tridimensional biomedical devices that bypass the skin barrier resulting in systemic and localized pharmacological effects. Historically, biomaterials such as carbohydrates, due to their physicochemical properties, have been used widely to fabricate MNs. Owing to their broad spectrum of functional groups, carbohydrates permit designing and engineering with tunable properties and functionalities. This has led the carbohydrate-based microarrays possessing the great potential to take a futuristic step in detecting, drug delivery, and retorting to biologicals. In this review, the crucial and extensive summary of carbohydrates such as hyaluronic acid, chitin, chitosan, chondroitin sulfate, cellulose, and starch has been discussed systematically, using PRISMA guidelines. It also discusses different approaches for drug delivery and the mechanical properties of biomaterial-based MNs, till date, progress has been achieved in clinical translation of carbohydrate-based MNs, and regulatory requirements for their commercialization. In conclusion, it describes a brief perspective on the future prospects of carbohydrate-based MNs referred to as the new class of topical drug delivery systems.


Introduction
Over the last few decades, we have witnessed captivating technology development in drug delivery systems. This technology improvement has enhanced the therapeutic efficiency of biotherapeutics (e.g., vaccines and macromolecules) and hydrophilic drugs [1]. The therapeutic efficiency depends on the administration route, the aim is to overcome the challenges of the conventional drug delivery system [2]. The notable drawbacks of the oral and parenteral delivery system are the first-pass metabolism in oral dosage form and poor patient compliance, respectively. This led to the need to develop a delivery system with reduced dosing frequency, self administration, and better patient compliance, economical and ecofriendly [3,4].
Emerging techniques, like the transdermal drug delivery system (TDDS), mainly focus on delivering therapeutic agents through the skin. Universally known, the transdermal route evades the first-pass metabolism, drug-food, and drug-drug interaction, thus enhancing the bioavailability of the therapeutic agents [5,6]. An ideal candidate for TDDS is a hydrophobic drug molecule with potent action, low molecular weight (M/W ≤ 400 Dalton), and lower diffusion rate. Though an attractive alternative, TDDS possesses a major hurdle-stratum corneum, i.e., the skin barrier [3,6].
A three-dimensional, needle-like biomedical device that efficiently delivers the drug through the skin barrier without disturbing and overcoming the conventional method's limitation, microneedles present themselves as a promising development for TDDS, comprising micronsize arrays organized in a small area [7,8]. This device is developed with the aim to overcome the shortcomings of a hypodermic needle and transdermal patch. The first research was published in the late 2000s, and since then, the research activities have grown exponentially, as shown in Fig. 1.
A systematic review and meta-analyses (Prisma) are an evidence-based minimum set of records for disclosure of systematic review and meta-analysis. The preferred reporting records emphasize the propagation of studies by analyzing randomly assigned records and can be utilized as a critical element for disclosing systematic reviews of different research types, predominantly estimating the intrusion. These guidelines are designed to assist authors to enhance the presentation of systematic review and meta-analysis and used for journal peer reviewers and editors for critical evaluation of the published systematic review. It also delivers comprehensive procedures relating to the technique, including eligibility criteria, information source, search strategy to execute a systematic review, and meta-analysis [9].
In the past, a wide range of materials were used for the fabrication of MNs. Initially, MNs were fabricated using silicon [10][11][12], titanium oxide [13], stainless steel [14], ceramics [15], and glass [8]. Polymers play a vital role in the fabrication of MNs and a key pillar for clinical applications [16,17]. Biopolymers, predominantly carbohydrates, were considered over synthetic polymers because of their biocompatibility, low toxicity, biometric characteristics, and biodegradable properties [18][19][20]. The parade of carbohydrates existing in nature has permitted the fabrication of MNs with exceptional features [18][19][20][21][22][23]. Several reviews on microneedles exist, but none exhaustively concentrated on carbohydrate-based MNs and reviewed using PRISMA guidelines [9]. Hence, considering the existing significance and the extent of carbohydrates, this review discussed challenges during the fabrication of carbohydrate-based MNs for drug delivery. Finally, it is highlighted with the prospect and clinical presence of carbohydrate-based MNs.

Eligibility criteria
A literature search was performed on English language papers using the PRISMA guideline [9]. Peer-reviewed articles published in the year January 2000-May 2020 were included, and publications before January 2000 were excluded. Studies describing carbohydrate-based MNs and studies related to the in vitro, in vivo data of carbohydratebased MNs were chosen to be included. Articles published only in the English language were included.

Search strategy
A search of the PubMed database identified studies published until May 2020. MEDLINE, Scopus, Google Scholar, Web of Science, and gray literature were incorporated into the search strategy. The search terms included 'microneedle drug delivery' and 'carbohydrate-based microneedle'. It also included terms like clinical practices, bioavailability, fabrication, microneedle array, and patient compliance.

Quality article and data analysis
The article quality and references of relevant articles were assessed individually according to the PRISMA protocol criteria. The quality criteria used to describe the studies were Fig. 1 Cumulative publications on the microneedle drug delivery system. The analysis was done by the PubMed database using the search term "microneedle drug delivery" [98] biopolymers, design, evaluation, and safety of the microneedle, preclinical, and clinical presence. A detailed description of the criteria used for the qualitative assessment of the records is given in Table 1.

Results of PRISMA analysis
A literature search was conducted using PRISMA guidelines. Search strategies were set with different keywords and specific assessment criteria for inclusion and exclusion of records, as shown in Fig. 2. As of 1190 records identified from prime research-comprehensive databases, 159 papers were retained to include the keyword "carbohydrate-based microneedle" in the search strategy. Fourteen records were excluded as they were formed in other languages and review articles to give 145 inclusions. According to the inclusion criteria given in Table 1, from the 145 inclusions, 15 papers were further excluded during full-text screening and data extraction as they did not describe carbohydratebased microneedles. Finally, a total of 130 records were considered for a systematic review.
Generally, MNs are categorized based on their assembly ( Fig. 4 (A)), design ( Fig. 4 (B)), and tip shape ( Fig. 4 (C)).  , accessible in many more forms [8,28]. Microneedle arrays are optimized by choosing the array dimensions and design, which might inflict less pain, good mechanical strength, and drug delivery [28]. Gills H.S investigated that a decrease in microneedle length and microarrays reduces pain and establishes that there is no role of microneedletip angle, thickness, and width in the degree of pain caused by the application of MNs [29].

Approaches for drug delivery with MNs
The delivery of drug-using MNs is characterized according to five different types and related forms of drug delivery (Fig. 5, Table 2).

Poke-and-patch approach
The first method is to poke the epidermis layer by microarray, which produces microchannels for drug delivery, subsequently detaching the microarray. Then, a transdermal patch is applied above it. Furthermore, to improve permeability, the electro field may be used. Solid microneedles follow this approach for the enhancement of skin permeability.

3.1.2.2
The coat-and-poke approach The second way is to coat the microneedle arrays with the drug dispersion. The coated drug dissolves after piercing the coated microneedles  into the skin. Microarrays can be covered with several compounds, including small molecules, proteins, plasmid DNA, virus particles, polymer nanoparticles, and water-soluble compounds.

3.1.2.3
The poke-and-release approach As a novel alternative to coated microneedles, the drug is encapsulated within microneedles made up of polymers and polysaccharides for rapid and sustained release in the skin. As a result, after piercing into the skin, the microneedles dissolve or degrade, releasing the encapsulated drug in a controlled manner.

3.1.2.4
The poke-and-flow approach In contrast to solid microneedles, hollow microneedles offer extra abilities and fewer difficulties. Hollow microneedles have complex fabrication processes with intrinsically weaker and sophisticated geometry. They permit the liquid formulations, which allow diffusion of the drug rapidly or with controlled flow rates, as required, but the rate-limiting step in hollow microarrays is the maintenance of flow rate.

Mechanical characterization of microneedles and safety evaluations
Mechanical characterization is a crucial step in the successful fabrication of MNs. Extensive stress during the insertion and removal of MNs is applied. Hence, it is essential to perform mechanical characterization and analyze the safety of MNs [11]. A variety of tests available to check mechanical strengths [30], are as given below.

Axial force mechanical test
In this test, force is applied to the MNs perpendicular to the base plate ( Fig. 6a) [31]. The applied force can be analyzed using a mechanical station, which details both displacement and force against a hard surface at a definite rate [32,33]. If a fracture occurs, a rapid drop can be seen in the force-displacement curve produced and considered as failure force [33]. A single microneedle (MN) is used to analyze failure force, which does not accurately simulate the pressures faced during insertion into the skin [30,34,35]. After piercing MNs into the skin, the forces are dispersed over a greater MN area as the flexible skin encases the MNs. The said axial force test is often used to assess polysaccharide MNs.   [33]. Again, a rapid reduction in the force-displacement curve shows the MN defect [31]. This test is carried out on a row of MNs rather than a single array, which calculates transverse fracture force per MN (Fig. 6b). According to the literature, this test's critical drawback is that the metal probe should be parallel with the MN array manually, which leads to inaccuracy [31,32].

Base-plate strength and flexibility tests
Assessment of base-plate strength is crucial as the abovementioned mechanical strength. Fracture of the base plates during piercing to the skin is not acceptable. Hence, base plates should be flexible enough to consent to the skin structure deprived of rupturing (Fig. 6c). A three-point bending test can be used to analyze base-plate strength. Donnelly et al. utilized a Texture analyzer to apply force with a metal probe to the base plate sitting between two aluminum blocks. Also, the base-plate flexibility is evaluated with the bending fracture of MNs [31].

Microneedles' safety
Microneedles are based on the principle of piercing the outermost skin layer, which is not similar to the conventional transdermal patch. The suitability of microneedles has been evaluated and predicted on the degree of adverse effects, namely, itching, scaling, prickling, stinging, tightness, burning, edema, and erythema. No one has reported carbohydrate microneedle-related local infection. The disturbance may occur during the piercing or compatibility of the material used for fabrication with skin or residual solvents in the needles. The microneedle is designed to avoid pain while piercing. A formed microchannel must be reclosed in the short term to eradicate the contamination. The microneedles and formulation should be biocompatible with the skin. Modepalli et al. studied the safety and toxicity of hydrogel-forming microneedles on the human skin. They stated no toxicity in cell lines due to the microneedle [37]. Sodium hyaluronate and polyvinyl pyrrolidone used to fabricate dissolving microneedles were examined for cytotoxicity using an MTT assay to assess cell viability of more than 87% at the end of 72 h. The biological safety of ibuprofen sodium-loaded hydrogel-forming microneedles was studied and observed that no bioburden was traced [16]. Drug-loaded hyaluronic acid microneedles led to no allergic or irritant-contact dermatitis, hence, considered as a safe product for human use [43]. In certain cases, MNs pierce the epidermis and dermis layer, generally sterile areas of the body [7]. Thus, MNs must not contain microbes that may lead to systemic infections. Also, microbial penetration through MN-created holes is significantly low than conventional hypodermic needle puncture. The possible solution may be the production of sterile MNs, but the sterilization method should be carefully considered to evade alteration of key features of the product and increase production cost. For instance, aseptic manufacturing will be costly, terminal sterilization with moist heat or microwave heating, and gamma radiation could damage the MNs. McCrudden et al. evaluated the effect of different sterilization methods on dissolving and hydrogel-forming MNs [16]. Thus, safety studies can be performed for the polymeric matrix which will be used for the fabrication of MNs.

Carbohydrate-based microneedles
Carbohydrates play a quintessential role in the fabrication of MNs, and thus form the emphasis of this review. Carbohydrates, such as sugars, hyaluronic acid [44,45], cellulose [46,47], and chemically modified methacrylate hyaluronic acid [48], carboxymethylcellulose [49,50], are utilized alone, in a blend, or as a composite. They are similar to the extracellular matrix and are simply recognized and accepted by the human body [51]. They are eliminated by metabolism and excretion through the renal system, evading tissue accretion [52]. Carbohydrates are approved by the US Food and Drug Administration (US FDA) and classified as generally recognized as safe (GRAS) [53,54].
Carbohydrates reveal an enormous structural disparity and several intrinsic characteristics, which impact the mechanical properties and invasion ability of MNs and influence the biological potency and durability of the therapeutic agent [55][56][57]. Certain traits, like the polymer's weight, swelling index, and an interface between the polymer and active agent, can stimulate and alter the drug-release rate [2,58,59]. The innumerable topographies impact their in vivo behavior and can be grouped into dissolvable (dextran, sodium chondroitin sulfate, and hyaluronic acid), biodegradable (chitin, starch), and swellable polymeric microarrays (methacrylate hyaluronic acid) as summarized in Table 4. The following section gives an overview of various carbohydrates. Table 5     The efficiency of drug administration using MNs was more effective than the oral route; these MNs could replace the current method for protein/peptide delivery [90] Dextran Poly-L-arginine Approximately, 75% of MNs dissolved after application in rat skin Efficacy of Drug depends upon dosage regimen, also can be a substitute to skin allergy device [130] described in the literature detailing their primary element, a therapeutic agent, and their biomedical applications. In a separate study, Chen et al. [66] fabricated Calmette-Guerin bacillus-loaded HA MNs, for tuberculosis prevention, which dissolved within 10-15 mins. As stated by the World Health Organization (WHO), the Calmette-Guerin bacillus vaccine is stable at 2-8°C. This research reported good stability at ambient temperature and quick onset of action for the undeveloped area. For nanomedicine delivery, Wang et al. [67] formulated pH-sensitive dextran nanoparticles loaded in cross-linked HA MNs to treat skin cancer. The nanoparticles comprised dextran derivative, alginate, glucose oxidase, and aPD1 in an acid-degradable polymeric matrix. When applied to the melanoma site, this microdevice converts glucose to gluconic acid, producing an acidic environment and subsequent antibody release.
Another study involved direct incorporation of cells and polymer, like the adipose-derived stromal vascular fraction cells carrying HA MNs, for diabetic wound healing. Cells loaded with HA MNs depicted improved wound healing compared with HA MNs and isolated sections, proposing an alternative approach [68]. In a different light, Park et al. designed amylopectin MNs to deliver essential nutrients, specifically niacin and ascorbic acid, wherein the amylopectin was used to enhance the mechanical strength of HA MNs in a ratio of 1:2.3 (HA:amylopectin). The higher concentration of amylopectin resulted in a stiffer and brittle structure, tough to remove from the mold without breaking. Dong et al. [69] formulated Au nanocages and doxorubicin-loaded HA MNs as Au nanocages reinforce the mechanical endurance and photothermal effect of a drug on exposure to infrared light to results in the eradication of exterior skin tumors.
In another approach to customizing drug delivery, Kim et al. [70] designed HA MNs with trehalose and polyvinylpyrrolidone to facilitate peptide delivery. The study implied that polyvinylpyrrolidone prevents peptide agglomeration and enzymatic degradation, thus enhancing the dissemination of the drug into the systemic circulation. MNs for disease diagnosis were also performed. Chang et al. [48] developed swellable MNs with methacrylate HA for metabolic detection, wherein the MN patch extracts sufficient interstitial fluid in a shorter time without the need of any external device.
Cross-linking of HA may cause swelling and tailor the degradation or drug delivery [59,71]. Zhang et al. [59] [72]. The incorporation of proteins and peptides was also studied. Monkare et al. developed HA MNs loaded with IgG, which recovered 82% of the protein with no change in the tertiary structure after fabrication [73]. Liu et al. studied the stability of fabricated insulin-loaded MNs at different temperatures for a month and reported MN stability, with more than 90% insulin detection. Insulin release from such MNs was enhanced after storage and resulted in a glycemiclevel reduction from 43 to 88% [74].
In different prospects, HA's feasibility was demonstrated by e-beam sterilization, which impeded the ascorbic acid 2glucoside degradation of HA MNs as the dissolution rate and drug release, structural features, and fracture force remained unaffected, without activity loss of capsulated active elements [70]. In vivo studies revealed that the release of the drug was achieved within 5 mins after insertion, and microarrays were completely dissolved within 15 mins in the skin [79].

Chondroitin sulfate MNs
On the other hand, Zaric et al. [80] fabricated human adenovirus type-5 vector-loaded carboxymethyl cellulose and sucrose (protein stabilizer) MNs for vaccine delivery. This research established the potential of MNs to sustain the antigenicity of attenuated vaccines after freeze-drying. Another study was performed on Lactobacillus-loaded carboxymethyl cellulose MNs, enhancing local skin health and rat's immune functions [80], while Lahiji et al. fabricated valproic acid-loaded carboxymethyl cellulose MNs to treat hair regrowth. Enhancement in the valproic acid release was observed and compared with the topical formulation, which elevates hair follicles' regrowth [81].

Chitin and chitosan MNs
Chitin and chitosan, after cellulose derivatives, are the widely used biopolymers produced by the deacetylation process. Chitosan comprises a high-density positive charge due to its acidic nature, allowing tissue and mucoadhesion [82][83][84]. Degradation of chitosan occurs by hydrolysis of acetylated residues, and its degradation rate is related to molecular mass and the degree of deacetylation [52]. Apart from these, the FDA has identified chitosan as GRAS, used for wound dressing and cartilage repair [54,85,86], and utilized for the fabrication of MNs for controlled and sustained drug delivery [85].
Jin et al. formulated chitin MNs coated with antigens, a convenient diagnostic tool for tuberculosis. Chitin MNs were water-insoluble, mechanically robust, physiologically inert, and exhibited a lower swelling index. Chen et al. [83] prepared chitosan MNs to allow the sustained release of bovine serum albumin (BSA). These chitosan MNs when pierced into porcine skin showed a 20% burst release of BSA after 30 mins. Subsequently, 95% of BSA was released within eight days. Within this period, chitosan MNs soften and break after insertion, which results in the retention of some polymeric pieces in the skin [83]. Besides this, Chen et al. [82] also developed tip-loaded chitosan MNs comprising model antigen ovalbumin for sustained delivery. These effective chitosan MNs contained biodegradable baking of poly (L-lactide-co-D, L-lactide), overcoming impression after piercing, which is not detected post 6 h. The in vivo study revealed that 50% of the antigen was released within the first two days, and a slight fluorescence was detected in three weeks [82].

Starch-based MNs
Starch is a biodegradable polymer extensively used due to its ease of processing and filmogenic ability for biomedical purposes [87]. While formulating, owing to its brittle behavior, starch is combined with other polymers [88]. Ling et al. substantially mixed starch with gelatine to transmit insulin, which loaded within 5 mins. Captivatingly, more than 90% of insulin was stable at ambient temperature for 30 days, specifying costeffectiveness and convenience after application [89].
Pullulan (PL), a hydrophilic biopolymer formed by the yeast-like fungus Aureobasidium pullulans in starch and sugar cultures, plays a crucial role in biomedical applications. This biopolymer has unique characteristics, such as oxygen-barrier property, mechanical robustness, chemically inert, transparent, and filmogenic ability with no toxicity or mutagenicity. Hence, PL is an outstanding candidate for the fabrication of dissolvable MNs. Vora et al. fabricated PL MNs to deliver proteins and peptides, namely FITC-BSA and insulin. Ex vivo studies demonstrate protein/peptides detected within 15 mins when applied to porcine skin [90].

Clinical presence and regulatory requirement for commercialization
Many nonclinical studies were conducted for carbohydratebased microneedles, which revealed enhancement in efficiency and permeability, but only a few accomplished positive clinical testing results. Table 6 summarizes active and completed clinical trials using carbohydrate-based microneedles [91]. Since microneedles are novel drug delivery systems, there are no particular requirements established to date. The conventional transdermal patch is simply applied to the skin's exterior surface, whereas MNs pierce the stratum corneum and even interrupt the epidermis and dermis. This mechanism motivates the necessity of regulatory demands. Henceforth, new provisions should be defined for the MN, apart from the existing transdermal patch. Some essential regulations that need to be considered with the commercialization of MN arrays are given below: 1. Materials, length, sharpness, needle characteristics, and geometry should be correctly designed 2. The MN arrays should retain satisfactory microbiological standards 3. The MN arrays should keep the uniformity of content 4. Quality manufacturing, safety, and packaging should be maintained 5. Safe and nonhazardous procedures should be drawn for MN arrays 6. A self-disabling mechanism assuring a single use may require the MN arrays to avoid reuse by patients or others. 7. If MN arrays are reusable, cleaning information should be provided 8. For long-term use, material safety should be considered with precision 9. Labeling should contain instructions for use 10. MN arrays should be used with a proper applicator for proper insertion and pain-free delivery. Also, arrays should give reproducible results without complications 11. Immunological safety assurance should be considered

Conclusion and future perspective
Drug delivery via the transdermal route has seen incredible development, among which the microneedle arrays facilitating drug infusion via forming several microchannels in the stratum corneum have grown exponentially over the past two decades. In the past fifteen years, the aspect of material science has transformed radically, and the utilization of natural polymers for recuperative purposes would be a critical success [53,92,93].
Carbohydrate usage presents an inclusive array of benefits owing to their intrinsic physiological compatibility, degradability, nontoxic, and sustainable nature. The inherent variation in carbohydrates, stemming primarily due to animal and plant origin, absence of standardization, and variation in appearance, with time or development, are few aspects that play a significant drawback role during commercialization [92]. The appropriate assessment and complete description of carbohydrates are necessary for analysis to overcome these drawbacks. Additionally, a significant feature emphasizes the probability of degeneration, decay, or adulteration of these resources during mining and development. The mechanical properties of carbohydrates can be enhanced by integrating with additional biopolymers [89], or nanosystem [69], and also cross-linking [94], which allows rupture of skin. The batch-to-batch variation impacts on mechanical qualities of a biopolymer.
Restructuring the study protocols as per the properties of the carbohydrates is essential. For instance, in moistureabsorbing polymers, assessments forecasting storage in water-resistant packages should be conducted. Along the same lines, considering the probabilities of utilizing these microarrays in states with high RH like the WHO climatic zone IV, deficiency of availability, and setups, appropriate contingencies should be planned [89]. The critical feature of MNs is the ease of self-administration. For bulk production and self-utilization, a unique applicator should be accompanied to allow adequate application, resulting in increased costs, but it is essential to ensure a preset force to assure skin insertion and its consequent advantages. Instability of some drug molecules to the fabrication step necessitates stabilizers into the polymeric formulation, thus stabilizing the drug during fabrication and long-term storage.
The actual application of MNs delayed as the regulatory guidelines are ongoing. Still, the academy is bringing novel instigating records in this zone, which highlights the difficulties during developments. The formation of policies to manufacture and analyze MNs is authoritative to ensure specifications and precision during production. Larraneta et al. [95] explained the need for specification for the analysis of MNs. The small MN tips are safe and minimally invasive. The critical principle of MNs is piercing the skin by generating microchannels, which reconcile after a few hours and release the active medicament [96]. However, the skin's microchannels may allow bacteria entry, though the probability of bacterial contamination is significantly lesser than conventional injection [97]. The incidence of infection can be reduced by applying an alcoholic solution before applying MNs [96]. Piercing of MNs provides a proinflammatory microenvironment and immunological effects when applied frequently. Thus, the efficacy of MNs can be regulated by the skin's mechanism. Currently, applicators are used to pierce the MN patch in the skin with a defined force. Variability can be seen when applied by a different candidate. The responsive biomarkers are used to assure that MNs are effectively applied.
Sterilization of the MNs is a crucial step that impacts the stability of the active pharmaceutical agent. Concerning the storage condition, drug-loaded MNs must be stable at ambient temperature for their intact mechanical properties and pharmacological performance. Another critical aspect emphasizes MN arrays' disposal, which may transfer blood-borne disease through blood or ISF [97]. Nanocarriers or nanostructured polymer loaded carbohydrate MNs are widely used for drug delivery and diagnosis. This technique can be a substitute for the conventional dosage. Nowadays, MNs can detect pH or change in temperature.
In conclusion, sophisticated design and impressive fusion of carbohydrate-based MNs effectively deliver several active pharmaceutical agents with a broad spectrum of applications in various zones. Carbohydratebased MNs have a fundamental part in wellness management, which provides stimulating revolutions in drug delivery. In these arrays, it is anticipated that such designs might be legalized in clinical practice, fetching motivation from nature into day-to-day drug delivery applications in a couple of years.
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