Cryogenic Microneedles: A Next-Gen Approach for Stable and Precise Dose Delivery

Over the decades, the idea and the concept of delivering the dosage form through the skin rather than into the skin has mainly evolved from fiction to clinical reality. The designed microneedle patches provide an array of pain-free injections that mainly breach the stratum corneum, which is often referred to as the body's toughest layer. Through these, medicines can be given effortlessly into the epidermis and the dermis, which eventually reach the immune cells, capillaries, and the targeted tissues while preventing the pain of injection in patients. However, the delicate molecules such as the mRNA, antibodies, enzymes, and living cells provide a major obstacle. They crumble under the excessive heat, pressure, and the solvents that are mainly used in the microneedle fabrications. so to overcome this, a fusion of cryogenics and micro-engineering works best, which is often referred to as the cryoMN. The technique of CryoMNs is mainly designed for frozen and biologically laden cryoprotective media that mainly form the ice-spikes that hold the life at zero stillness.1 When it is applied by pressure to the skin, the ice tips that pierce the barrier melt quickly and release the dosage form. Mostly, they signal a new era where the frozen precision meets the transdermal delivery system.

Design and fabrication

The cryoMNs are mainly fabricated by filling the microneedle molds with the cryogenic medium, which is mainly composed of an aqueous solution with cryoprotectants such as trehalose, glycerol, or DMSO. Then these moulds are frozen so that they form a rigid and needle-like structure that can be effectively demolded as a patch. The tip of the frozen needle mainly maintains the rigidity at the cold temperature for insertion into the skin, and then it slowly dissolves in situ at the viable skin temperature. It also contains various variants like the frozen hydrogels, frozen solutions, etc.2

Fig 01: concept of cryoMN

Materials and the cryoprotectants –

The formulation is mainly selected based on the needle mechanical strength, which is mainly required at the time of insertion, the cryoprotection, and the compatibility with the excipients. The commonly reported components mainly used are the sugar protectants such as sucrose and trehalose, PEG, hyaluronic acid, and a low concentration of DMSO.3 For manufacturing the cell-containing cryoMNs, a higher grade of cryoprotectants and controlled freezing is essential. The recent advances in technology use the hydrogel precursors that mainly freeze into the mechanically robust cryo-tips, and eventually they melt to release the cargo. 4

Fabrication of the cryoMNs –

The fabrication method is compatible and straightforward with the existing micromolding workflow that is-

However, the industrial scale-up requires a close and concise attention to produce the freezing rates, aseptic handling of the system, and the cold chain for storing and transporting. Mainly, the automated filling and the controlled freezers have been used to improve the batch uniformity for clinics. 5

Fig 02: fabrication of CryoMNs.

Mechanism of action: release profile, penetration into the skin, and preservation.

The cryoMNs are a unique fusion of engineering and the cryogenic technique. unlike the traditional needle, which mainly swells the matrices processed at an elevated temperature, the cryoMNs employ the rigid tip freezing, which is capable of penetrating the stratum corneum and the epidermis with less trauma. Once it gets inserted, the frozen matrix melts rapidly or even dissolves upon contact with the tissues, which have a warm temperature. Then the encapsulated payload is directly released into the epidermis of the dermis layer, where the antigen-presenting cells, which are mainly the Langerhans and the dendritic cells, are concentrated.6 This unique temperature-triggered delivery system ensures the spontaneous release and the localization of the payloads, which include mainly the proteins, nucleic acids, and living cells. These sensitive agents are mainly stored in a frozen, cryoprotective environment. The cryoMNs effectively lower the thermal and the enzymatic degradation before their delivery. Comparing the room-temperature polymeric microneedles that mainly require solvent processing, crosslinking, and drying, the cryoMNs eventually maintain the original conformation and the biological activity of the biomolecules.7 The nucleic acid therapeutics, mainly the cryogenic storage with an optimized buffer, which is mainly enriched with sugars or glycol, protect against the attack of nuclease and the structural degradation. When this is applied to the living cell embedded cryoprotectants, they mainly preserve the cell viability and the immunogenicity function. The recent advances in the field of the cryoMNs have demonstrated the successful delivery of the tumor cell vaccine, which has indirectly provided the enhanced antitumor activity. 8

Preclinical applications and evidence –

1. mRNA and nucleic acids –

The intradermal delivery of luciferase mRNA using the cryoMNs mainly showed that the preserved mRNA integrity during molding and the functional protein expressed in the skin after the application. The use of cryoMNs allowed the direct loading of naked or formulated mRNA without exposure to high temperatures. This has led to decentralized mRNA vaccine patches and therapeutics wherein the conditions mostly favour cold temperatures. 9

2. Antibodies and protein biologics-

The reports suggest that cryoMNs can be used for the fragile and sensitive biologics and antibody fragments that mainly tolerate freezing with appropriate excipients. The antigen + immune checkpoint inhibitors have been shown in animal models to elicit an immune response, which mainly demonstrated that this technique can be effectively used for immunotherapies. 10

3. Vaccine and immunotherapy –

CryoMNs target the skin APCs for immune priming with the smallest antigen doses. The recent preclinical vaccine studies indicated that the antigens can be delivered intradermally, and the cryoMNs present the advantage of temperature-sensitive vaccines. 11

Advantages over the conventional microneedles

• Preservation of bioactivity – this technique mainly minimizes the denaturation and aggregation before the delivery of the drug.12
• Combinational therapy- the CryoMNs have the potential to enable co-delivery of the cells and the immune modulators in the models.13
• Minimally invasive- CryoMNs avoid the pain of hypodermic needles and mainly target the skin immune environment. 14

• Payload compatibility- CryoMNs mainly accommodate fragile antibodies that are incompatible with high-temperature processing.

Fig-03: Comparison Chart

Fig 04: Comparison Table

Limitations and challenges –

Though they provide a promising approach in the healthcare system, there are a few drawbacks to this dosage form, too-

• Cold- chain and storage conditions-

CryoMNs currently require cold storage that is -20 to -80°C or at least refrigerated. this mainly complicates distribution with the fully thermostable microneedle. With the use of lyophilized or freeze-dried microneedles and excipient improvement, the cold chain needs are relaxed, but don’t fully provide an alternative. 15

• Sterility and aseptic manufacturing –15

The manufacturing of the CroMNs requires an aseptic processing along with good manufacturing practice conditions. This mainly increases the cost and complexity of the dissolving patches. The regulatory pathways for cell-containing devices that are combined with a drug and the device framework they mainly require dedicated studies.16

• Mechanical strength –

The tips of CryoMNs are rigid at low temperatures but can be brittle. This ensures that there is consistent penetration with the fracture of the tip. But the storage and the transfer of these may cause its fracture of the tips. Hence the materials engineering can improve the fracture and provide resistance.

Future scope –

The research directions paved the way for the future directions of the CryoMNs. the development in this field would be a good alternative to the current microneedles. Some of the key future scopes are-

• Formulation development to reduce the dependence on cold chains.
• Use of mechanical engineering for tougher cryo-matrices that retain the insertion capability after the withdrawal. 16
• In situ freezing- this approach permits autologous cell loading at the care point

Fig 05: Future Scope