Formulation and Evaluation of Microneedle Based Transdermal Drug Delivery System by Using Antihypertensive Drug

1.1. Transdermal drug delivery system:

Definition – Transdermal drug delivery system is defined as self-contained, self-discrete dosage forms, which when applied to the intact skin deliver the drug at a controlled rate to the systemic circulation. ^(1,2) Transdermal drug delivery (TDD) is a route of drug delivery for treating or preventing disease by absorbing drugs through the skin, permeating into the skin and further into the blood circulation. TDD avoids first-pass effects, prolongs the action of drugs with short half-lives through slow release and avoids fluctuations in blood levels, reduces side effects and improves patient compliance. The stratum corneum barrier plays a key role in TDD, and many methods have been used to improve the efficiency of TDD, including the use of chemical penetration enhancers and different physical enhancement approaches, such as micro-needling, iontophoresis, electroporation, laser ablation and ultrasound facilitation. ⁽³⁾

1. 2.  Microneedle Based Transdermal Drug Delivery Systems:

In recent years, microneedles have gained widespread interest in TDD and have shown Brilliant achievements in delivering both chemical small molecules and biomacromolecules whilst being minimally invasive and painless. ^(4,5) Microneedles usually consist of micrometer-sized needles (50–900 µm in length) in the form of microneedle arrays that can successfully penetrate. the stratum corneum and deliver drugs in a minimally invasive manner below the stratum corneum without damaging blood vessels and nerves in the dermis ^(6,7), improving patient compliance and allowing drugs exposed in the epidermis or dermis to be rapidly absorbed by surrounding capillaries and lymph nodes. ^(8-10) Microneedles or Micro-needle patches or Microarray patches are micron-scaled medical devices used to administer vaccines, drugs, and other therapeutic agents.⁽¹¹⁾ While microneedles were initially explored for transdermal drug delivery applications, their use has been extended for the intraocular, vaginal, transungual, cardiac, vascular, gastrointestinal, and intracochlear delivery of drugs.⁽¹²⁾ Microneedles are constructed through various methods, usually involving photolithographic processes or micro moulding. These methods involve etching microscopic structure into resin or silicon in order to cast microneedles. Microneedles are made from a variety of material ranging from silicon, titanium, stainless steel, and polymers. Some microneedles are made of a drug to be delivered to the body but are shaped into a needle so they will penetrate the skin. Stimuli-responsive microneedles are advanced devices that respond to environmental triggers such as temperature, pH, or light to release therapeutic agents. The arrays are applied to the skin of patients and are given time to allow for the effective administration of drugs. Microneedles are an easier method for physicians as they require less training to apply and because they are not as hazardous as other needles, making the administration of drugs to patients safer and less painful while also avoiding some of the drawbacks of using other forms of drug delivery, such as risk of infection, production of hazardous waste, or cost. ⁽¹³⁾

1.3. Types of Microneedles:

As aforementioned, a microneedle is a micron-sized needle with a height of 10–2000 µm, and width of 10–50 µm, and are widely used in transdermal drug delivery systems to the advantage of safe, painless, convenient, non-invasive and efficient drug delivery.50- 51 Microneedles can create pores on the skin and enable the drug to penetrate through the epidermis layer to the dermal tissue directly. Unlike regular hypodermic needles, the microneedle has the ability to improve patient compliance as it does not hurt nerves. What is more, the microneedle delivery system delivers drugs transdermally, hereby improving the bioavailability of drugs via avoiding hepatic first pass metabolism. The drug delivery mechanisms of these microneedles (respectively) are the “poke and patch” approach, the “coat and poke” approach, the “poke and release” approach, and the “poke and flow” approach. We will subsequently introduce each type of microneedle in more detail, along with examples of successful use cases of each. ^(14,17) Morphologically, microneedles are classified into five types namely:

1. Solid microneedles
2. Coated microneedles
3. Dissolving microneedles
4. Hollow microneedles
5. Hydrogel-Forming microneedles.

1.4. Advantages of Microneedle Patch:

1. Minimally invasive
2. Pain-free
3. Enhanced drug absorption
4. Reduced dosing frequency
5. Improved patient compliance
6. Large molecules can be administered
7. Painless administration of the active pharmaceutical Ingredient
8. First-pass metabolism is avoided
9. Faster healing at injection site than with a hypodermic needle,
10. Decreased microbial penetration as compared with a hypodermic needle, the microneedle punctures only the epidermis
11. Specific skin area can be targeted for desired drug delivery enhanced drug efficacy may result in dose reduction
12. Good tolerability without long-term oedema or erythema
13. Rapid drug delivery can be achieved by coupling the microneedles with other technologies. ⁽¹⁸⁾

1.5. Disadvantages of Microneedle:

1. Careful use of the device may be needed to avoid particles ‘bouncing off’ the skin surface.
2. The thickness of the stratum cornea and other skin layers varies between individuals and so penetration depth of particles could vary too.
3. The external environment, like hydration of the skin, could affect delivery.
4. Repetitive injection may collapse the veins.
5. The tip of the microneedle may break off and remain within the skin on removal of the patch. ⁽¹⁹⁾

1.6. Material Compositions of Microneedles:

Microneedles can be made from various different materials, each yielding different characteristics and these materials can be used to prepare different kinds of microneedles via different methods. ⁽²⁰⁾ The materials of microneedles can be divided into various types:

1. Metal materials
2. Inorganic materials
3. Polymer materials
4. Glass materials
5. Ceramic materials.

1.7. Various Methodologies/Techniques:

There are Various Methodologies/Techniques involved in Microneedles Applications is as follows:

1.7.a. Poke with patch or poke and flow approach:

It involves pressing of solid microneedles array on the skin to produce microspores followed by the application of patch or liquid formulation containing therapeutic agent. The microspores formed allow the easy passage of drug in to the deeper tissues of the skin.

1.7.b. Coat and poke approach:

In this method, microneedle array is first coated with drug and then inserted into skin. Upon insertion, the drug-coated on the microneedles dissolves in the aqueous pores followed by permeation in to the surrounding skin tissues and microcirculation.

1.7.c. Scratch and patch approach:

It is a variation of poke and patch approach, where microneedle array is scraped over the skin and then applying patch or liquid formulation.

1.7.d. Dip and scrape:

In this approach, microneedles are first dipped into drug solution and then scraped across the skin surface to leave behind the drug within micro abrasions created by the needles.

1.7.e. Poke and release approach:

In this approach, the drug can be encapsulated within soluble sugar or biodegradable polymeric microneedles, followed by the insertion into the skin to achieve either fast or controlled drug release. Poke with flow: In this approach, hollow microneedle array is pressed on the skin to create micro-channels in the skin followed by infusion of liquid formulations in to deeper skin tissues across the needle pores. ⁽²¹⁾

2. Preformulation Studies:

Preformulation studies are investigations of the physical and chemical properties of a timolol maleate, such as organoleptic properties, solubility, melting point, and compatibility with polymer by using FTIR spectroscopy. Analytical method development: A simple, accurate, and precise UV-visible spectrophotometric method was developed and validated for the quantitative estimation of timolol maleate in accordance with ICH guidelines. The goal is to generate data that informs formulation design and development, resulting in a stable, safe, and effective drug product.

2.1. Fabrication of microneedle patch:

For formulation of microneedle patch Polymers like PVA, PVP K30, HPMC E4M, HPMC K4M, Plasticizers like Glycerin, PEG 400 and drug (Timolol maleate) was used. Aqueous blend of polymer and drug were used to fabricate microneedle patches by using different plasticizers with constant volume, as Specified in (Table no. 1 & 2). by observing each formulation, the best formulation batch was optimized.

Table No.1: Formulation of timolol maleate loaded microneedle patches by using Glycerin as plasticizer

Table No. 2: Formulation of timolol maleate loaded microneedle patches by using PEG 400 as plasticizer.

86.5 mg of timolol maleate and varying amounts of polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP K30) in ration 9:1 were dissolved in same volumes of distilled water and ethanol in ration 1:1 by keeping glycerin amount constant (as shown in table no. 3), then applying gentle heat using water bath. Distilled water and ethanol was selected as solvent owing to better solubility of drug in it. These polymer matrix solutions were transferred into ABS micromolds, and allowed to dry in vacuum desiccator for 24 h. After 24 h, microneedle patches were retrieved from micromolds and preserved in a moisture resistant container along with silica.

Table No.3: Formulation of Microneedle patch by increasing concentration of PVA: PVP K30 (9:1)

In above table the concentration of polymer (PVA: PVP K30) was taken in ratio 9:1, Because the other Ration like (8:2, 1:1, 2:8) didn’t have that kind of features like Mechanical strength of needles.

3. Evaluation of Microneedle Patch:

3.1. Physical Characteristics of Microneedle Patch:

After microneedle patch preparation the formulations were determined by visual examination for appearance, colour, shape, texture, and surface integrity.

3.2. Thickness Testing: ⁽²²⁾

Figure.1: Franz Diffusion Cell

3.8. Determination of permeation coefficient, flux and Lag time: ⁽²⁶⁾

The permeation studies were carried out on optimized microneedle patch batch is F2, modified Franz diffusion cell were used in permeation study. The assembly was kept on a magnetic stirrer and content of the receptor compartment was stirred with a magnetic bead at 500 rpm at the temperature of 37±1ºC. The samples were withdrawn (1 ml) at different time intervals and replaced with an equal fresh volume of phosphate buffer pH 7.4 saline. The collected samples were subjected to UV-Visible spectrophotometer analysis at 294 nm against pH 7.4 phosphate buffer for data analysis: Ex vivo permeation study data were plotted as cumulative amount permeation per cm² vs time. Several parameters such as a steady state drug flux (Jss), a permeability coefficient (Kp) through the microneedle patch, lag time (T lag) and within the membrane, linear curve taken as a drug flux (Jss) and its X-intersect took as lag time (T lag) and permeability coefficient (Kp) were resolved.

3.9. In vitro drug release kinetic study: ^(27-29)

To determine the drug release mechanism and to compare the release profile differences among microneedle patch gel formulations, the data obtained from the drug released amount and time were used. The drug release kinetics was analyzed with mathematical models. The three parameters were used to study the release mechanism i.e. release rate constant (k), correlation coefficient (R), and release exponent (n) and determine the best fit model for optimized formulation. The release data was analyzed with the following mathematical models:

3.9.a. Zero order kinetics:

Drug dissolution from pharmaceutical dosage forms that do not disaggregate and release the drug slowly (assuming that area does not change and no equilibrium conditions are obtained) can be presented by the following equation:

Q=K0t…                                                             Eqn (1)

3.9.b. First order kinetics:

This model has also been used to describe absorption and/or elimination of some drugs, although it is difficult to conceptualize this mechanism on a theoretical basis.

Q1=Qe-K1 t or Log QI=Log Q0+ Kit 2.303        Eqn (2)

3.9.c. Higuchi matrix model:

This model is used to study the release of water soluble and low soluble drugs incorporated in semisolid and/or solid matrices. Mathematical expressions were obtained for drug particles dispersed in a uniform matrix behaving as the diffusion media. It describes drug release as a diffusion process based on the Fick's law, square root time dependent.

Q=KH t1/2…                                                      Eqn (3)

3.9.d. Korsmeyer-peppas model:

Korsmeyer developed a simple, empirical model, relating exponentially the drug release to the elapsed time (t)

Ft or Mt/ M∞ =a. tn                                            Eqn (4)

3.9.e. Hixson-Crowell model:

Hixson and Crowell (1931) recognized that the particles regular area is proportional to the cube root of its volume. They derived the equation:

W0 1/3-Wt 1/3= k t                                             Eqn (5)

3.10. Stability studies: ^(30-34)

Stability of a drug has been defined as the ability of a particular formulation in a specific container to remain within its physical, chemical, therapeutic and toxicological specifications. A drug formulation is said to be stable if it fulfills the following requirements:

• It should contain at least 90% of the stated active ingredient.
• It should contain effective concentration of added preservatives, if any.
• It should not exhibit discoloration or precipitation, not develops foul odour.
• It should not develop irritation or toxicity.

The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity, light and enables recommended storage conditions, re-test periods and self-lives to be established.

RESULTS AND DISCUSSION:

4.1. Preformulation study:

Organoleptic Properties: Colour – White,

Odour – Odourless, Appearance - Crystalline Powder.

Identification of Timolol maleate has the same physical description properties as given in IP.

4.1.a.  Determination of Melting point:

The melting point of Timolol maleate was measured in the laboratory and found to be 202.5°C.

4.1.b. Determination of Solubility:

The solubility of the pure drug in 10mg/10ml of solvent was carried out and it reveals that it was soluble in water, 95% soluble in ethanol, partially insoluble in diethyl ether, and sparingly soluble in chloroform.

4.1.c. Fourier Transform Infrared Spectroscopy for Analysis of Drug and Polymer:

Graph no.1: IR Spectrum of Timolol maleate

Graph no.2: IR Spectrum of PVA

Graph no.3: IR Spectrum of Physical mixture of Timolol maleate & PVA

FTIR spectra of the Drug, Polymer & Physical mixture of both were shown in Graph no.1, 2, & 3. It was observed that, the principal peak was found in the FTIR spectra of a drug, & polymer, as well as the FTIR spectra of a physical mixture of drugs, and polymer.

4.1.d. Differential Scanning Calorimeter (DSC):

Graph no.4: DSC of Timolol Maleate (API)

The DSC of Timolol Maleate (drug) shows a sharp endothermic peak at 203.41°C, confirming its crystalline nature and corresponding to its melting point. (Shown in Graph no.4)

Graph no.5: DSC of PVA (Polymer)

The PVA polymer shows a broad endothermic peak at 193.95°C, with earlier thermal transitions around 46.5°C, indicating its semi-crystalline nature and thermal degradation behavior. (Shown in Graph no.5)

Graph no.6: DSC of Physical mixture API & Polymer (1: 1)

In the physical mixture (drug + polymer), the drug’s melting peak shifts to 206.55°C with reduced enthalpy and a broadening of the peak, suggesting a possible interaction or partial amorphization of the drug in the polymer matrix. Additionally, polymer-related transitions are still visible, indicating the presence of both components. Overall, the shift and decreased intensity of the drug peak in the mixture suggest potential miscibility or molecular interaction between Timolol Maleate and PVA. (Shown in Graph no.6). There is no physical and chemical interaction between drugs and polymers.

4.1.e. Spectrophotometric method for the estimation of Timolol Maleate:

Standard calibration curve of Timolol Maleate in 7.4 pH buffer:

From the standard curve, it was observed that the drug obeys Beer’s law in the concentration range of 1-10 µg/ml in phosphate buffer of pH 7.4. The drug showed good linearity with regression of coefficient (R² = 0.998) and the equation for this line obtained was found to be y= 0.0439x+0.0077, which is used for the calculation of amount of drug and dissolution study.

Graph no.7: Standard Calibration curve of Timolol maleate

4.2. Preparation of Micromold:

Dimensions & material used for Preparation of Micromold are given as follows:

• Shape: Circular
• Diameter: 3cm / 30mm
• Needle Height: 0.7mm – 0.8mm
• Needle density: 100 /cm²
• Needle tip: 30°
• Space between needles: 700um – 800um
• Patch thickness: 400um – 500um
• Material: ABS (Acrylonitrile butadiene styrene)
• Process: 3D Stereolithography Process

4.3. Preliminary trials for formulation of Microneedle patch:

4.3.a. Preparation of Microneedle patch by increasing Polymer concentrations:

Table no.4: Preparattion of Placebo batches of Microneedle patch by increasing concentration of Polymer

Note: Each Polymers like PVA, PVA: PVP K30 (9:1), HPMC E4M and HPMC K4M are used. The concentration of polymer (PVA: PVP K30) was taken in ratio 9:1, Because the other Ration like (8:2, 1:1, 2:8) didn’t have that kind of features like Mechanical strength of needles. In above table concentration of Plasticizer (i.e. Glycerin or PEG 400) was kept constant but the concentration of Polymer (PVA, PVA: PVP K30, HPMC E4M & HPMC K4M) was increases from 200 mg – 1000 mg and processed with solvent evaporator method.

4.3.b. Formulation of Microneedle patch by Increasing Plasticizers concentrations:

Table no.5: Preparattion of Placebo batches of microneedle patch by Increasing Concentration of Glycerin

Table no.6: Preparattion of Placebo batches of microneedle patch by Increasing Concentration of PEG 400.

n above tables concentration of Polymer (PVA, PVA: PVP K30, HPMC E4M & HPMC K4M) was kept constant but the concentration of Plasticizer (i.e. Glycerin or PEG 400) was increases from 10% - 50% based on weight of polymer taken in previous study, and processed with solvent evaporator method. From overall study on concentration of Polymer and Plasticizer, Batches with 200 mg Polymer (PVA, PVA: PVP K30, HPMC E4M & HPMC K4M) & 10% Plasticizer (Glycerin & PEG 400) batches was optimized as a better formulation than other batches, Formulation study was based on the above performed study.

4.4. Evaluation of Microneedle Patches:

4.4.a. Physical Characteristics of Microneedle Patch:

• Color: Transparent, some are slightly milky / cloudy.
• Surface & Texture: Smooth backing layer & Microneedle array on other side.
• Surface integrity: (Mentioned in SEM results).

4.4.b. Thickness Testing:

Thickness testing of prepared Microneedle patch was done by Gauge Vernier Caliper Thickness tester and the results are as follows:

Table no.7: Results for thickness testing of prepared Microneedle patch

The thickness testing results of the prepared microneedle patches, as shown in above table, indicate a considerable variation among different batches, with values ranging from 210 µm to 400 µm.

4.4.c. Weight Variations:

Table no.8: Results for Weight variations of prepared Microneedle patch

In above table which presents the weight variations of different batches (F1 to F8) of prepared microneedle patches, revealing significant inconsistencies in individual weights and corresponding percentage deviations. The ideal consistency is not maintained across batches, with deviations ranging from 22.6 % (F1) to +36.47 % (F2), indicating substantial under- and over-weighting issues. The substantial deviation in batches such as F1 and F2 highlights the need for stricter manufacturing control and optimization of the formulation or process parameters to achieve uniformity in weight, which is critical for ensuring dose accuracy and therapeutic efficacy.

4.4.d. Moisture content:

Table no.9: Results for Moisture content present in Microneedle patch

The moisture content results for microneedle patches in above table indicates significant variation across different formulations. Formulation F1 exhibited the highest moisture content at 3.61%, which could be attributed to the polymer (PVA) and the active pharmaceutical ingredient (Timolol maleate) combination potentially retaining more water. The maximum moisture content was found in formulation F1 which was prepared from polymer PVA & API (Timolol maleate).

4.4.e. Drug Content:

Table no.10: Results for Drug content present in Microneedle patch

The data presented in above table highlights the drug content percentages in various microneedle patch formulations (F1 to F8), with formulation F2 showing the highest drug content at 88.13%. This suggests that F2 had the most efficient drug-loading capability, likely due to an optimal combination or method used for incorporating Timolol maleate with the polymer PVA. The maximum drug content was found in formulation F2 which was prepared from polymer PVA & API (Timolol maleate).

4.4.f. Scanning Electron Microscopy (SEM):

According to Literature review microneedle has sharp needle but due to the Poor material of Micromold (ABS: Acrylonitrile Butadiene Styrene) which was affected when it come in contact with acetone, it was start to dissolve and because of it the patch also affected and the SEM results are not really good as expected. (See Figure no.2)

Figure no.2: Picture of Microneedle patch formulation captured during SEM

During SEM dimensions of Needles present on Microneedle patch are found to be as follows:

• Needle density: 220 needles per cm²
• Space between needles: 300um – 320um
• Height of needles: 90um – 120um
• Base width of needle: 275um – 320um

4.4.g. In vitro release study:

Drug release through microneedle patches, The Drug Release of formulation F1 = 6.98 %, F2 = 9.86 %, F3 = 8.82 %, & F4 = 8.51 %. F2 batch shows high drug release as compared to other batches after 12 hrs. Thus batch F2 is optimized.

Graph no.8: Drug Release (mg) Vs Time (hr)

4.4.h. Cumulative Drug Release

The cumulative percent drug release of formulation F1, F2, F3 & F4 was found to be 70.5 %, 98.6 %, 88.5 %, & 90.5 %, after 12 hrs. F2 Batch shows highest drug release as compared to other batches after 12 hrs. Thus, Batch F2 is optimized batch.

Graph no.9: Percent Cumulative Drug Release (%) Vs time (hr)

4.4.i. Determination of permeation coefficient, flux and Lag time:

Area of Franz Diffusion Cell = 1.76 cm², Slop = 0.1743

• Flux (Jss ) = Jss is the steady state slope of line and the slope of graph cumulative amount per cm² Vs time (hr). Calculated as 0.1743 mg/ cm²/min.
• Permeation coefficient (Kp) = Jss / Cs = 0.1743/1.66 = 0.104 cm/min
• Lag time = 120 min (2hr)

Note: Lag time was determined from graph of Cumulative amount permeation per cm² Vs Time (hr) by intercepting the straight line from 4^th point of graph to the x- axis and it was determined as 120 min (2 hr).

Graph no.10: Cumulative amount permeation per cm2 Vs Time (hr)

4.4.j. In vitro drug release kinetic study:

Table no.11: Kinetics of drug release of F2 Formulation

Graph no.11: Zero order release kinetics

Graph no.12: First order release kinetic

Graph no.13: Higuchi Matrix Model

Graph no.14: Korsmeyer Peppas Model

Graph no.15: Hixson- Crowell Model

From the above Graphs, the release kinetics data indicate that the drug release from the formulation best fits the zero-order model (R² = 0.9878), suggesting a constant drug release rate over time, independent of drug concentration. This is ideal for maintaining steady therapeutic levels. The Hixson-Crowell (R² = 0.9316) and Higuchi (R² = 0.9204) models also show good correlation, implying that drug release may be influenced by changes in surface area and diffusion mechanisms, respectively. The Korsmeyer-Peppas model (R² = 0.7913) shows moderate fit, indicating a potential combination of diffusion and erosion-controlled release, while the first-order model (R² = 0.7026) shows the least correlation, suggesting that the release is not primarily concentration-dependent. Overall, the dominant release mechanism appears to be zero-order, highlighting the controlled and sustained nature of drug release from the system.

4.4.k. Stability studies:

Table no.12: Results of Stability study of Microneedle patch at different storage conditions