INTRODUCTION:

During recent years, transdermal drug delivery systems have shown a tremendous potential for their ever-increasing role in health care. The transdermal technology have limitations due to the inability of a large majority of drugs to cross the skin at the desired therapeutic rates because of the presence of a relatively impermeable thick outer stratum corneum layer. This barrier posed by human skin limits transdermal delivery only to lipophilic, low molecular weight potent drugs^[^1]. When oral administration of drugs is not feasible due to poor drug absorption or enzymatic degradation in the gastrointestinal tract or liver, injection using a painful hypodermic needle is the most common alternative. An approach that is more appealing to patients, and offers the possibility of controlled release over time, is drug delivery across the skin using a patch.

However, transdermal delivery is severely limited by the inability of the large majority of drugs to cross skin at therapeutic rates due to the great barrier imposed by skin’s outer stratum corneum layer ^[2].

Chemical means include the prodrug approach and/or use of chemical penetration enhancers that can improve the lipophilicity, and the consequent bioavailability. On the other hand, physical means of transdermal drug delivery comprises of iontophoresis, electroporation, and sonophoresis. Chemical approach increases the lipophilicity and therefore increase the permeability of drugs across skin, where as the physical approaches disrupt the upper layers of skin the stratum corneum(SC) and reduce the resistance to the passage of drugs by creating minute holes in the skin that are large enough for the passage of smaller drug molecules but probably small enough not to damage the skin. Microneedle technology has been developed as an advanced technique for penetration of large molecular weight and/or hydrophilic compounds. Micron scale needles assembled on a transdermal patch have been proposed as a hybrid between hypodermic needles and transdermal patches to overcome the individual limitations of both the injections as well as patches. Microneedle technique has been successfully used to deliver a variety of compounds including macromolecules and hydrophilic drugs into the skin. As microneedle system bypasses the stratum corneum barrier of the skin, permeability enhancement of two to four orders of magnitude has been observed for small molecules like calcein and also for the relatively larger compounds like proteins and nanoparticles^[^1].

THE SKIN:

The skin is the largest organ of the body ^[3,4], accounting for more than 10% of body mass, and the one that enables the body to interact more intimately with its environment. The skin consists of 4 layers. The SC, the outer layer of the skin (nonviable epidermis), forms the main barrier for diffusion for almost all compounds. It is composed of dead, flattened, keratin-rich cells, the corneocytes. These dense cells are surrounded by a complex mixture of intercellular lipids, namely, ceramides, free fatty acids, cholesterol, and cholesterol sulfate. Their most important feature is that they are structured as ordered bilayer arrays. The predominant diffusional path for a molecule crossing the SC appears to be intercellular ^[5,6]. The other layers are the remaining layers of the epidermis (viable epidermis), the dermis, and the subcutaneous tissues (Figure 1). Associated appendages include hair follicles, sweat ducts, apocrine glands, and nails. In a general context, the skin’s functions may be classified as protecting, maintaining homeostasis, and sensing ^[7]. Many agents are applied to the skin either deliberately or accidentally, with either beneficial or deleterious outcomes. The main interest in dermal absorption assessment is related to (a) local effects in dermatology (eg, corticosteroids for dermatitis); (b) transport through the skin to achieve a systemic effect (eg, nicotine patches, fentanyl patches); (c) surface effects (eg, sunscreens, cosmetics, and anti-infective); (d) targeting of deeper tissues (eg, nonsteroidal anti-inflammatory agents); and (e) unwanted absorption (eg, solvents in the workplace, pesticides, or allergens). The skin is popular as a potential site for systemic drug delivery, on the one hand because of the possibility of avoiding the problems of stomach emptying, pH effects, enzyme deactivation associated with gastrointestinal passage, and hepatic first-pass metabolism.

ADVANTAGES AND DISADVANTAGES OF MICRONEEDLES:

Advantages ^[8]:

· Using microneedles reduces the chances of pain, infection, and injury.

· In many cases, microneedles lead to greater patient satisfaction.

· Microneedles can be fabricated to be long enough to penetrate the SC but short enough not to puncture nerve endings.

· Frequent dosage is not required.

· Using microneedles avoids first-pass effect.

· Microneedles allow rapid penetration of drugs into the systemic circulation.

· Administration of drugs via microneedles bypasses the gastrointestinal tract.

· Microneedles can provide direct controlled delivery of small molecules, macromolecules, vaccines, or nucleic acids into the viable epidermis.

· A relatively large surface area can be treated.

· Single-use needles are easily disposable and potentially biodegradable.

· The technique is minimally invasive.

· Drug can be administered at constant rate for a longer period.

· By fabricating these needles on a silicon substrate because of their small size, thousands of needles can be fabricated on single water. This leads to high accuracy, good reproducibility, and a moderate fabrication cost.

· Hollow microneedles could be used to remove fluid from the body for analysis such as blood glucose measurements and to then supply microliter volumes of insulin or other drug as required.

· Hollow like hypodermic needle; solid increase permeability by poking holes in skin, rub drug over area, or coat needles with drug.

Disadvantages ^[8]:

· The needles are very small and much thinner than the diameter of hair, so the microneedle tips can be broken off and left under the skin.

· Microneedles can be difficult to apply on the skin; the clinician must learn proper application technique.

· Skin irritation may result because of allergy or sensitive skin.

· Local inflammation may result if the amount of drug is high under the skin.

NEED OF USING MICRONEEDLE:

When oral administration of drugs is not feasible due to poor drug absorption or enzymatic degradation in the gastrointestinal tract or liver, injection using a painful hypodermic needle is the most common alternative. An approach that is more appealing to patients, and offers the possibility of controlled release over time, is drug delivery across the skin using a patch ^[9]. However, transdermal delivery is severely limited by the inability of the large majority of drugs to cross skin at therapeutic rates due to the great barrier imposed by skin's outer stratum corneum layer. To increase skin permeability, a number of different approaches has been studied, ranging from chemical/lipid enhancers ^[10] to electric fields employing iontophoresis and electroporation^[^11,12] to pressure waves generated by ultrasound or photo acoustic effects^[13,14]. Although the mechanisms are all different, these methods share the common goal to disrupt stratum corneum structure in order to create “holes” big enough for molecules to pass through. The size of disruptions generated by each of these methods is believed to be of nanometer dimensions, which are large enough to permit transport of small drugs and, in some cases, macromolecules, but probably small enough to prevent causing damage of clinical significance. An alternative approach involves creating larger transport pathways of micron dimensions using arrays of microscopic needles. These pathways are orders of magnitude bigger than molecular dimensions and, therefore, should readily permit transport of macromolecules, as well as possibly supramolecular complexes and microparticles. Despite their very large size relative to drug dimensions, on a clinical length scale they remain small. Although safety studies need to be performed, it is proposed that micron-scale holes in the skin are likely to be safe, given that they are smaller than holes made by hypodermic needles or minor skin abrasions encountered in daily life. Transdermal drug delivery is a noninvasive, user-friendly delivery method for therapeutics. However, its clinical use has found limited application due to the remarkable barrier properties of the outermost layer of skin, SC. Physical and chemical methods have been developed to overcome this barrier and enhance the transdermal delivery of drugs. One of such techniques was the use of microneedles to temporarily compromise the skin barrier layer. This method combines the advantages of conventional injection needles and transdermal patches while minimizing their disadvantages. As compared to hypodermic needle injection, microneedles can provide a minimally invasive means of painless delivery of therapeutic molecules through the skin barrier with precision and convenience. The microneedles seldom cause infection while they can allow drugs or nanoparticles to permeate through the skin. Increased microneedle-assisted transdermal delivery has been demonstrated for a variety of compounds. For instance, the flux of small compounds like calcein, diclofenac methyl nicotinate was increased by microneedle arrays. In addition, microneedles also have been tested to increase the flux of permeation for large compounds like fluorescein isothiocynate-labeled Dextran, bovine serum albumin, insulin and plasmid DNA and nanospheres.

MECHANISM:

The mechanism of action depends on the microneedle design and is summarized in (Figure 2). All types of microneedles are typically fabricated as an array of up to hundreds of microneedles over a base substrate. Solid microneedles can be either pressed onto the skin or scraped on the skin to create microscopic holes, thereby increasing skin permeability by up to 4 orders of magnitude. This is followed by application of drugs or vaccines from a patch or topical formulation. Residual holes after microneedle removal measure microns in size and have a life time of more than a day when kept under occlusion but less than 2 hours when left uncovered. The second strategy is to have vaccines or drugs encapsulated in a dry coating onto solid microneedles ^[15]. This coating can dissolve within 1 minute after insertion into skin, after which the microneedles can be withdrawn and discarded. As an alternative to insoluble metal or polymer microneedles, complete microneedles have been fabricated out of biodegradable or water-soluble polymers. Model drugs have been encapsulated within polylactic co-glycolic acid (PLGA) microneedles for controlled release over hours to months^[^16] and, more recently, within water-soluble carboxymethyl-cellulose, polyvinyl-pyrrolidine, and maltose for rapid release within minutes. The final approach consists of using hollow microneedles to puncture the skin followed by infusion of liquid formulation through the needle bores in a manner similar to hypodermic needle.^[17,18]

MATERIALS AND METHODS:

The materials used for fabrications are ^[19]:-

· Silicon

· Metal

· Polymers

· Glass

Silicon: First the microneedles were fabricated using silicon to have sharp and hard microneedle because of greater mechanical strength. Fabrication by microneedle is costly because it require clean room microfabrication for processing. Silicon is brittle and may break in the skin.

Metal: Metal are used for fabrication as they have good mechanical strength, cost is low, metal used are stainless steel, titanium, nickel, iron.

Glass: Fabrication is also done by glass. They are physically capable of insertion into the tissue and they have high drug loading capacity and one can see how much amount of drug is delivered after use.

Polymers: Biodegradable polymer used are Polylactic acid and Polyglycolic acid because they are cost effective. This biodegradable polymer is used owing to the chance of microneedle breaking off in the skin.

METHODOLGY FOR DRUG DELIVERY

A number of delivery strategies have been employed to use the microneedles for transdermal drug delivery. These include

· Poke with patch approach

· Coat and poke approach

· Biodegradable microneedles

· Hollow microneedles

· Dip and scrape

Poke with patch approach^[^20]:

It involves piercing an array of solid microneedles into the skin followed by application of the drug patch at the treated site. Transport of drug across skin can occur by diffusion or possibly by iontophoresis if an electric field is applied.

Eg: Insulin Delivery

Coat and poke approach ^[20]:

In this approach needles are first coated with the drug and then inserted into the skin for drug release by dissolution. The entire drug to be delivered is coated on the needle itself.

Eg: Protein vaccine delivery

Biodegradable microneedles ^[20]:

It involves encapsulating the drug within the biodegradable, polymeric microneedles, followed by the insertion into the skin for a controlled drug release.

Hollow microneedles^[^20]:

It involves injecting the drug through the needle with a hollow bore. This approach is more reminiscent (suggestive of) of an injection than a patch.

Eg: Insulin Delivery

Dip and scrape ^[20]:

Dip and scrape approach, where microneedles are first dipped into a drug solution and then scraped across the skin surface to leave behind the drug within the microabrasions created by the needles. The arrays were dipped into a solution of drug and scraped multiple times across the skin of mice in vivo to create microabrasions. Unlike microneedles used previously, this study used blunt-tipped microneedles measuring 50–200 μm in length over a 1 cm² area.

eg: DNA Vaccine Delivery

TYPES OF MICRONEEDL

Classification depends upon the fabrication process:-

· Solid microneedles

· Hollow microneedles.

Solid Microneedles:

· Solid microneedle are fabricated in 750-1000μm in length 15-20⁰ tappered tip angle.

· Increase the permeability by pocking the holes in skin, rub drug over area or coat needle with drug.

· Wear time is short, ranging from 30 seconds to 10 minutes.

Hollow microneedles:

· Hollow microneedles are fabricated with lumen diameter 30μm and height 250μm.

· Used continuously to carry the drug in to the body by diffusion.

· Large amount of drug are delivered to obtain therapeutic effect.

· Hollow microneedle is also used to remove the fluid from the body for analysis.

FABRICATION OF MICRONEEDLE:

Microneedles can be fabricated employing microelectro mechanical systems (MEMS). The basic process can be divided in to three parts: deposition, patterning and etching.

Microneedles have become a new type of the bio-medicine injector, it can throw the cuticle and not excite the nerve, and the patient will feel nothing. Moreover, it can be made by different kind of materials, like as SU-8, PMMA, PDMS, COC, silicon etc.

Deposition: Refers to the formation of thin films with a thickness anywhere between a few nanometers to about 100 micrometers.

Patterning: Is the transfer of a pattern onto the film.

Lithography: Is used to transfer a pattern into a photosensitive material by selective exposure to a radiation source such as light. This process can involve photolithography, electron beam lithography, ion beam lithography or X-ray lithography. Diamond patterning is also an option for lithography.

Etching: Is a process of using strong acid or mordant to cut into the unprotected parts of a material’s surface to create a design in it and can be divided into two categories: wet etching or dry etching. The selection of any of the above mentioned methods largely depends on the material of construction and the type of microneedle.

Polymer Microneedles^[^22]:

The SU-8 2050 as the material of the microneedles. SU-8 is a high contrast, epoxy based photoresist designed for micromachining and other microelectronic applications. The UV-light absorptivity of SU-8 is lower than others and therefore can get the high-aspect ratio structures. As the turning model technique to fabricate is the microneedles, V-groove on the silicon wafer should be considered. At first, the V-groove is built with the depth of 320 μm on silicon wafer, and then coated with the SU-8 on the silicon wafer with the depth of 450 μm and use the scraper to put the SU-8 uniformly. Silicon wafer covered the SU-8 will be set in the oven to break away the bubbles and to smooth the surface by heating and vacuum. Finally, the SU-8 developer is used to form the microneedles.

The series of fabrication processes contain the several steps as below and shown in Fig. 4:

1) Wafer cleaning: Mixing H₂SO₄ with H₂O₂ as the Piranha to clean the silicon wafer and heat it up at 150 °C to remove the mist.

2) Depositing the oxide and coating the photoresist: The depths of oxide and photoresist are 1 μm and 4 μm respectively.

3) Photolithographic: Utilizing the first mask to develop the V-groove.

4) Remove the oxide and photoresist: Using the BOE and Acetone to remove the oxide and photoresist separately.

5) KOH wet-etching: The KOH solution is 30 wt% and the temperature is set at 60°C.

6) Coating the negative photoresist, SU-8: The thickness of SU-8 is 450 μm.

7) The 2^nd photolithographic: This process develops the position of the bottom of microneedles.

8) Departing the SU-8 from the silicon wafer: Put the silicon wafer in the MF-319 and the microneedles will move off the silicon wafer.

Fabrication of silicon microneedle^[^22]:

1) Cleaning the wafer to remove the hydrocarbon and the mist.

2) Depositing the Oxide and Nitride with the depths are 1 and 0.2 μm respectively.

3) Coating the photoresist, AZ-4620.

4) Photolithographic and develop the microneedles.

5) Remove the unprotected Oxide and Nitride by Reactive Ion Etching, RIE.

6) KOH wet-etching: The KOH solution is also 30 wt% at 80°C. As shown in (Figure 5)

EVALUATION OF MIRONEEDLES:

Margin of safety ^[23]:

Forvi et al. defined the margin of safety as the ratio between the force required for piercing the stratum corneum and the force at which microneedles broke. They hypothesized that if the ratio is <1 then microneedle array can be used in biomedical application. They checked margin of safety for silicon microneedles using computerized apparatus. For compressive failure force measurement, Enduratec station was used in which microneedles were placed between punch and load cell. An appropriate margin of safety was found for sample silicon microneedle arrays.

Measurement of fracture force ^[23]:

The force required for mechanical fracture of a microneedle was tested by Davis et al., employing an axial load test station that drove the microneedle against a flat block of aluminium at a rate of 0.01 mm/s until a preset displacement of 500 mm was reached. Microneedles were attached to the testing surface using adhesive tape around the base of the needle. Microneedle fracture was observed through an attached microscope to evaluate the mode of failure. The force and displacement data were used to quantitatively determine the fracture force.

Measurement of insertion force into human skin ^[23]:

A displacement–force test station was used by Shawgo et al. to measure the force applied to a needle, needle position and skin resistance during the sequence of the needle’s translation, deflection of tissue around the needle and insertion into the skin of human subjects. A drop in electrical resistance of the skin was used to identify needle penetration since visual observation of needle insertion was extremely difficult. The electrical resistance of skin’s outermost layer, the SC, is much greater than deeper tissues, therefore the resistance of the skin drops dramatically as soon as a needle penetrates.

Biological safety test:

Wu et al. determined extractable chemicals from microneedles. Extraction of chemicals from microneedles was done by immersing microneedles in physiological saline at 37°C for 72 h. The extract was then directly applied on shaved intact human skin for checking dermal irritation. Negative result of the test revealed the biological safety of the microneedles.

Penetration/diffusion test:

In-vitro and ex-vivo test:

In-vitro/ex-vivo tests are performed on isolated animal/ human dermatomed skin to study penetration or diffusion of drug from a dosage form to its site of application. These tests can also be used to compare the depth of penetration of the molecule. Wu et al. used confocal laser scanning microscopy (CLSM) to demonstrate the depth of penetration of Rhodamine B in human dermatomed skin using microneedles of 150 mm length. They reported the concentration of the dye to be very weak below 80 mm depth. They also evaluated the penetration of model drug using Franz diffusion cell across the microneedle-treated and untreated skin and reported enhancement in penetration by 10⁴to 10⁵ times with use of microneedles. Similar observations were also reported by other researchers. Paik et al. investigated penetration of microneedles both in vitro and ex vivo by injecting Rhodamine B dye. For in-vitro testing 1% agarose gel was used and for ex-vivo testing, chicken breast flesh, laboratory mouse and an anaesthetized rabbit were used. The Rhodamine B easily penetrated across the 1% agarose gel and chicken breast flesh; the penetration in 1% agarose gel can clearly be viewed in (Figure 6).

Functional capacity test^[^23]:

Wang et al. evaluated the functional capacity of micro fluidic lumens using a custom fluidic test set up. The test setup consisted of a syringe pump system with a dye-filled syringe, a polymer tube and microneedle array. This syringe pump system was used to examine the formation of the microneedle lumens by allowing dye to flow from the syringe to the microneedle orifice. Microscopic inspection of the microneedle tips and the base plate during the micro fluidic characterization can be used to detect cracks in the base plate and passage continuity.

Characterization of microneedle geometry^[^23]:

Scanning electron microscopy can be used to determine the base radius, tip radius and wall thickness of the microneedles. Interfacial area (i.e. the effective area of contact) between the needle and the skin) can be calculated in two ways:

(1) the annular surface area,Aa; at the needle tip

A_a₌π(r_tt-t²/4) ------1

and (2) the full cross-sectional area, Af; at the needle tip

A_f₌πr_t² -----2

Needle wall angle, a, is calculated as

α = tan^-1(r_b_–r_t/h)

Where r_t is the outer radius of the microneedle tip, r_b is the outer radius at the needle base, t is the wall thickness and h is the height.

APPLICATIONS:

Insulin delivery ^[1]:

Insulin is one of the most challenging drug of all times for the drug delivery technologists. Martano et al, used micro arrays for the delivery of insulin to diabetic hairless rats. Solid microneedles of stainless steel having 1mm length and tip width of 75 μm were inserted into the rat skin and delivered insulin using poke with patch approach. Over a period of 4 hours, blood glucose level steadily decreased by as much as 80% with the decrease in glucose level being dependent on the insulin concentration.

DNA vaccine delivery ^[2]:

The cells of Langerhans present in the skin serve as the first level of immune defense of the body to the pathogens invading from the environment. These cells locate the antigens from the pathogens and present them to T lymphocytes, which in turn stimulate the production of antibodies. Mikszta et al reported the delivery of a DNA vaccine using microneedle technology prepared with the dip and scrape approach. The arrays were dipped into a solution of DNA and scrapped multiple times across the skin of mice in vivo. Expression of luciferase reporter gene was increased by 2800 fold using microenhancer arrays. In addition, microneedle delivery induced immune responses were stronger and less variable compared to that induced by the hypodermic injections.

Desmopressin delivery ^[20]:

M. Cormier et al (Alza Corporation, USA) examined the use of microneedles to deliver desmopressin, a potent peptide hormone used in the treatment of nocturnal enuresis in young children, as well as for the treatment of diabetes insipidus and haemophilia A. Microneedles were coated by an aqueous film coating of desmopressin acetate on titanium microneedles of length 200 μm, a maximal width of 170 μm and a thickness of 35 μm. Microneedle patch was inserted into the skin with the help of an impact applicator. A target dose of 20 μg of desmopressin was delivered to hairless guinea pig from 2 cm²microneedle array within 15 minutes.

Protein vaccine delivery ^[2]:

Matriano et al. examined the use of microneedles to deliver ovalbumin as a model protein antigen coated onto the needle surface. Microneedles were prepared with a dry-film coating of antigen and then inserted into the skin of hairless guinea pigs in vivo using a high velocity injector. Insertion depth was shown to average 100 Am, with 300 Am as the maximum depth. A range of doses was given by varying the antigen solution concentration coated onto the needles and the number of needles used. Antigen release from the needle surface was found to occur quickly, where up to 20 Ag could be released within 5s. Using a prime-plus-boost protocol, antibody responses were found to be similar for microneedle delivery and intradermal injection, and up to 50-fold greater than subcutaneous or intramuscular injection of the same antigen doses. The greater immune responses to the two intracutaneous delivery methods is proposed to be caused by the presence of antigen presenting Langerhans cells in the basal epidermis. Coating microneedles with both antigen and a glucosaminyl muramyl dipeptide adjuvant increased antibody responses further. A related study also demonstrated delivery from microneedles coated with desmopressin. The microneedle arrays (i.e. microprojections) used in this study were acid-etched from a titanium sheet and measured 330 Am in length. Array size was either 1 or 2 cm² with a needle density of 190 needles per cm². The significance of this study is that it demonstrates protein antigen delivery to generate an antibody response using microneedles. It also establishes the feasibility of using a dry-coat method to deliver compounds from microneedles.

In Transdermal Drug Delivery ^[21]:

The success of transdermal has been severly limited by the inability of most drugs to enter the skin at therapeutically useful rates because the stratum corneum does not have any nerves since microneedles that are long enough and robust enough to penetrate across this layer, but short enough to not stimulate the nerves in the deeper tissue, have the potential to make transdermal delivery a painless and much more viable option with use of hollow microneedle it allows the delivery of medicine, insulin, proteins or nanoparticles that would encapsulate a drug or demonstrate the ability to deliver a virus for vaccination. An array of needle ranging from 300-400 needles can be designed to puncture the skin and deliver the drug.

Figure 1. Schematic representation of the skin layers

Fig 2: Schematic representation of different microneedle designs for transdermal drug delivery: A) solid microneedles, B) solid microneedles coated with dry drugs, C) polymeric microneedles with encapsulated drug or vaccine and D) hollow microneedles for injection of drug solution

Fig 3: Hollow microneedle

Solid microneedle

Fig:4 Fabrication process of polymer microneedle