Accepted Manuscript

Kinetics of collagen microneedle drug delivery system

Abhilash Aditya, Beomjin Kim, Rina D. Koyani, Beu Oropeza, Michael Furth, Jihye
Kim, Namsoo Peter Kim

PII: S1773-2247(18)31052-9
DOI: https://doi.org/10.1016/j.jddst.2019.03.007
Reference: JDDST 970

To appear in: Journal of Drug Delivery Science and Technology

Received Date: 13 September 2018

Revised Date: 28 February 2019
Accepted Date: 9 March 2019

Please cite this article as: A. Aditya, B. Kim, R.D. Koyani, B. Oropeza, M. Furth, J. Kim, N.P. Kim,
Kinetics of collagen microneedle drug delivery system, Journal of Drug Delivery Science and Technology
(2019), doi: https://doi.org/10.1016/j.jddst.2019.03.007.

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Kinetics of Collagen Microneedle Drug Delivery System

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Abhilash Aditya, 1Beomjin Kim, 1,2Rina D Koyani, 1Beu Oropeza, 1Michael Furth, 1Jihye Kim
and *1Namsoo Peter Kim

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1
Department of Metallurgical Materials and Biomedical Engineering (MMBME), The University of
Texas at El Paso, TX, USA.

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Current position: School of Pharmacy, The University of Texas at El Paso, TX, USA.
*Corresponding author: nkim@utep.edu; Tel: +1 915-747-7996; fax: +1 915-747-8036

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Abstract
Microneedle fabrication using biopolymers has emerged as a potential technique to offer
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additional advantages of biodegradability and enhanced biocompatibility over other polymers.
Collagen, a biopolymeric source of extracellular support in human skin, serves the purpose of
adaptation and synthesis of dissolvable microneedles as well. Hence the present study is
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designed to synthesize dissolvable collagen microneedles using micro-molding technique

whereas Taguchi analysis was employed to evaluate their correlations and conclude the
outcomes as stabilized conditions to produce uniform microneedles. The drug loading and
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releasing potential of microneedles were demonstrated using trypan blue as a model drug.
Moreover, in vitro penetration of fabricated microneedles was conducted on mice skin and
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histological imaging witnessed its practical potential. This collagen based dissolving
microneedles may serve as the promising device for delivering collagen as well as other drugs
for the betterment of skin and other health issues respectively, in no time.
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Key words: Collagen, Microneedles, Biopolymer

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1. Introduction

Among all four modes of microneedles (MNs) introduced as a replacement of hypodermic

injections i.e. solid MNs [1], hollow MNs [2], coated [3], and dissolving MNs [4], the dissolving
MNs has attracted more attention of researchers, and the need for a better dissolving needle

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spurred the use of biopolymers. Biopolymeric microneedle architecture has broadened the
prospective for biodegradable, biocompatible, economic, simple, and reliable transdermal drug
delivery system. Previous studies designed for dissolvable microneedles, using a range of

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polymers like maltose [5], carboxymethylcellulose or amylopectin [6], dextrin, chondroitin
sulfate and albumin [7] etc. have been exploited for the delivery of proteins, enzymes, serum,
pharmaceutical materials and other needed pharmaceutical uses. Dissolving microneedles are

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also found appealing for vaccination where it enhances the immunogenicity by targeting the
antigen delivery to skin [8]. Encapsulation of bioactive molecules to these water-soluble
microneedles and their successful delivery to skin is a remarkable offering [6,7]. Moreover the

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logistic advantage of eliminating the risk of biohazardous wastes makes them an attractive
proposition for an eco-friendly drug administration system.
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Chitosan, gelatin, poly lactic acid (PLA), polyvinyl pyrrolidone (PVP), poly vinyl alcohol (PVA),
Poly (L-lactide-co-glycolide; PLGA) etc. are widely utilized for the fabrication of biopolymeric
dissolvable microneedles [8,9,10], however collagen has not been explored utterly for the same
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due to its nature. Even though collagen is tough and robust material reinforcing the skin, tooth,
and bone strength, the limiting factors of the collagen strength, deformation of collagen
microfibrils, and the origins of toughness remain largely unknown [11]. The amount of collagen
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in the skin can also be a responsible for aging [12] and elevation of collagen level in the skin can
improve the skin aging. The artificial technique used for the same is called Collagen induction
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therapy. However it is also accompanied with limitations of blood exposure and requirement of
complete anesthesia of the skin while performing [13]. Hence, the present study is an effort to
alter the conventional method and introducing new possible technique to deliver collagen
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through skin using dissolving collagen microneedles.

Collagen is endowed with highly biodegradable, biocompatible, non-toxic and weakly antigenic
nature than many other biopolymers and hence offers an excellent answer to many limitations
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[14]. Ensuring the employment of the inherent properties of this biopolymer, the study was
conducted for the synthesis of dissolving collagen microneedles with auxiliary support of other
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biopolymer i.e. polyvinyl pyrrolidone (PVP) in order elevate the strength of the microneedles to
form and sustain the geometrical dimensions of the microneedles. PVP being a strong
biopolymer, supports the collagen to retain the dimension. Specific attention was paid to the
process parameters suitable for synthesis of dissolving collagen microneedles proposing
improved dimensional configurations. The statistical outcome demonstrates the logistic layout to
produce collagen - PVP microneedles. Moreover, the drug loading and release efficiency of the
microneedles were also demonstrated using trypan blue as a model drug, and its penetration
potential was measured by skin histology studies.
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2. Materials and methods

2.1 Materials

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Hydrolyzed collagen was purchased from Perfect Hydrolyzed Collagen Peptides, Perfect
Supplements (LLC, USA) and polyvinylpyrrolidone with molecular weight K-90 (111.14g/mol)
was derived from Making Cosmetics, Snoqualmie, Washington, USA. Trypan blue, Phosphate

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buffer and NaCl were purchased from sigma Aldrich. However, Polydimethylsiloxane (PDMS)
microneedle molds were procured from Micropoint Technologies (Micropoint Technologies Pvt.
Ltd, Singapore). The mice skin used in experiments were kindly provided by Department of

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Biosciences, The University of Texas at El Paso. All the other chemicals used in the study are of
analytical grade.

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2.2 Microneedle preparation and optimization of processing parameter
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Polydimethylsiloxane (PDMS) microneedle molds were filled with the biopolymer mix
prepared with hydrolyzed collagen and polyvinylpyrrolidone with molecular weight K-90
(111.14g/mol) at different W/V ratio. Microneedle patches of 10×10 arrays were prepared with
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height to base ratio of 300:100, 400:150, and 500:200 (µm) respectively. Micromolds filled with
biopolymer mix were placed inside a vacuum oven for desired formulation. Optimization of
processing parameters (i.e. control factors) such as microneedle height (300, 400 and 500 µm),
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pressure (0.02MPa, 0.04MPa and 0.06MPa below atmospheric pressure), time (12, 18 and 24
hours), and the concentration of collagen and PVP in distilled water (8%, 10% and 12% weight
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ratio) was conducted to check their influence on dimensions and structure of the microneedles.
Once the microneedle patches are dried, they were carefully removed from the molds.
Synthesized microneedles with all the control parameters were photographed using optical
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microscope (Nikon AZ 100 multi-zoom optical microscope, University of Texas at El Paso,

USA) followed by image processing and analyzing software for measuring the heights of the
microneedles (Figure 2).
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2.3 Statistical Analysis

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Influence of processing parameters (control factors) on the microneedle formation

(performing parameters) has been evaluated statistically using Taguchi method, specifically L9
Orthogonal Array method consist of 3 levels and 4 variables. Whereas the height, pressure drop,
duration and the concentration are the four control factors and each control factor has 3 different
levels (Table 1). L9 Orthogonal Array experimental set up has been laid out as exhibited in Table
2 and essential data has been derived through set of calculations performed as mentioned below:
• The volume ratio is the ratio of actual volume to ideal volume of the microneedles which
was calculated using the formula for volume of square pyramid because the shape of
microneedle is square pyramid (equation 1). The ideal volumes (vi) of each microneedle
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with height 300µm, 400µm and 500µm were 1µm3, 2µm3 and 6.67µm3 respectively,
while derivation of the actual volume (va) accommodated in the microneedles was
subjected by measuring height and base of synthesized microneedles through microscopy
(Figure 2).

-------------------[Equation 1]

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Where,
V: volume

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h: Height of the microneedle

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b: base of the square pyramid shaped microneedle

•
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The modulus of deviation of the actual and ideal volume ratio is said to be the
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performance parameter, as calculated using equation 2. The performance parameters
are dependent on the control factors and hence calculated and noted for every
parameter separately.
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-------------[Equation 2]
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• However, the ranking of control factors was determined based on the signal to noise ratio
(S/N) graph, which was calculated as equation 3.
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------------- [Equation 3]

Where, X: Mean
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SD: Standard Deviation

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2.4 Dye release rate from dissolving microneedles

Micromolds were filled with a mixture of 60µl of trypan blue dye (0.4% liquid, Sigma
Aldrich, USA) and 60µl collagen – PVP solution and kept inside the vacuum oven for 3 hours
for dye penetration into the microneedle holes at the molds and dry completely. The molds are
then removed from the vacuum oven and excess dye was removed leaving behind the dye filled
molds and left at room temperature to dry and solidify. Then the solution of collagen and PVP
was added into the molds and kept inside the vacuum oven for 12 hours to prepare the bed and
ultimately the dissolving collagen microneedle patch carrying trypan blue (Figure 1).
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Prepared microneedles were subjected to dye release study in two different liquid
mediums i.e. distilled water (DI) and 0.8% NaCl. The microneedle patches were placed in liquid
medium and measured for the dye release at every 20 seconds using spectrophotometer
(BioMateTM 3S). Concentration of released trypan blue was determined using Trypan Blue
standard graph and release rate was determined as Figure 4. However the loading profile of
Trypan blue was determined by sum of the dye concentrations released at every 20 seconds

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during release rate experiment, which exhibits the total actual amount of trypan blue dye loaded
in each size of microneedles (Figure 5).
2.5 Histological Studies

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The 400µm collagen microneedle is pierced on to the mouse skin and the tissue samples
were processed using a Thermo Scientific Spin Tissue Processor Microm STP-120. Tissue

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dehydration was done by immersing tissue samples in different concentration of ethanol (starting
with 70%, 95%, 100%), followed by immersion in xylene two times and paraffin infiltration.
Paraffin embedded tissues were sectioned to 6µm Shandon Finesse ® E/ME microtome.

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All samples were deparaffinized in three xylene washes then rehydrated in decreasing
concentrations of ethanol (100%, 95%, 70%, 50%) with each wash lasting 3 min. Hematoxylin
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and eosin (H&E) staining was done post-deparaffinization. Following rehydration, the samples
were exposed to hematoxylin solution for 2 minutes then rinsed with running water for 1-minute,
distilled water for 30 seconds and 95% alcohol for an additional 30 seconds. Immediately after
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the samples were exposed to eosin solution for 1 minute then washed with increasing amounts of
alcohol (95% x2, 100% x2) for 2 minutes each then two xylene washes each for 2 minutes. The
processed samples were then micro-photographed using an optical microscope (Nikon AZ 100
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multi-zoom optical microscope, University of Texas at El Paso, USA).

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3. Results and Discussion

3.1 Fabrication of collagen based microneedles, Influence of processing parameters on

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microneedle synthesis and Statistical analysis

The latest approach of synthesizing dissolvable microneedle using biopolymers can offer
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better applicable attitude over non-dissolvable and non-biopolymeric systems. Following weak
biopolymers like carboxymethyl-cellulose or sugar, strong biopolymers such as PVP, PVA, PLA,
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and PLGA have gained comprehensive attention owing to advantages offered over the previous
once. Number of proposed studies of microneedle formation for creating effective transdermal
drug administrative systems were based on utilizing these biopolymers [10, 14, 15]. However,
ductile nature of collagen categorized it among the weak biopolymers while comes to application
but inherent beneficial properties of collagen have contributed to its proven record of safety and
efficiency as a carrier for pharmaceutical drugs, proteins and biomolecules [16,17,18]. For the
present investigation, we explored inexpensive and non-complex micro mold technique for the
synthesis of collagen-based microneedles. The mitigation of the same required additional
strengthening prop and use of PVP served the purpose of aiding concrete base enhancing the
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strength and increasing durability of the microneedles. To achieve desirable characteristics of

microneedles, blend of these two polymers (collagen: PVP) were optimized for the most
appropriate concentration where as 8% (W/V) and 7:3 (Collagen: PVP) ratio was found to
project the consistent microneedle formation (Figure 2). However, increase in PVP concentration
enhances the viscosity of solution, making it difficult to penetrate the mold cavities, and
lowering the PVP concentration weakening the micro-mold strength. Viscosity of the

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formulations also influence the drug loading and delivery efficiency of the microneedles; a
higher viscosity boosts the drug loading [19]. As collagen is the main biopolymer that is
expected to be delivered, PVP content is minimized to certain level.

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Beside concentration, design parameter such as height of the microneedles is also known
to influence the microneedle efficacy [20]. Mechanical strength of microneedles also depends on
the aspect ratio of the microneedle i.e. cross-sectional area at the same base width/diameter [6].

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Apparently, upsurge in height elevates the drug cargo loading to microneedles, nevertheless
longer microneedles can be sensitive to break with force and can cause pain to patients as well
[21]. Effect of microneedle design on pain in human subjects Furthermore, height of the

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microneedles also effects the rate of the drug flux [15], and so desirable height for controlled
skin penetration and drug delivery is indeed. In present investigation, three different heights i.e.
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300, 400 and 500 µm were analyzed for their impact to develop mechanically stable
microneedles (Figure 2) and correlate its impact on volume lodging.
The success of any drug delivery system relies on their ability to hold the drug in its most
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appropriate form and deliver it with same formula. Henceforth processing conditions play the
most vital role where preventing deleterious effect of high temperature and pressure denaturing
the protein or drug is indispensable. The mild processing environment provide the benefits of
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encapsulating any biomolecule sensitive to exacting conditions and perseverance of incorporated

characteristics of encapsulated fragile biomolecule. The maintained temperature for present
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study was 25° C which is in fact utterly suitable for any kind of drug or biomolecule, whereas
pressure and time were the considerable variable. Smooth penetration of fluid formulation in
microchannels of mold also relies on the pressure drop in addition to microneedle geometry,
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fluid viscosity and density [22] and the time required for drying embedded micro-molds is in
direct correlation with temperature and pressure set.
To understand the dependence and influence of control parameters on the processing
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parameter i.e. volume ratio accommodated in the synthesized microneedle, Taguchi analysis was
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implicated.
According to the response table (Table 3), the plot for S/N ratio (Figure 3) points out that the
microneedle height has the most statistical significance on the microneedle dimension. 300µm
microneedles are with remarkable uniformity across the array and is most influential among
other parameters. This is because longer microneedles are more prone to damage when
synthesized in square pyramid shape. The duration of the microneedle (time) which it spent
inside the vacuum oven is the second most influencing factor after the microneedle height. The
contribution of each control factor is shown illustrated by performing ANOVA for the
performance parameter (modulus of deviation of volume ratios) (Table 4).
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The error and noise are null for this setup. The effect of the pressure drop is low and is
assumed to be due to the error. Therefore, this parameter is considered as error and F ratio is
calculated as shown in Table 4. Hence, the microneedle height and the time spent by the
microneedle inside the vacuum oven are statistically significant to this build at the given
confidence interval of α = 0.15.
From all the experiments conducted with different variables it is evident that the

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microneedles with 300 microns height and the time of 12 hours in vacuum oven gives the
optimistic results and are very close to perfect dimensions. Maintaining all the favorable
conditions, the resultant microneedles derived were mechanically robust and sharp with

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pronounced piercing potential, and hence offers the non-complex and specific fabrication process
for manufacturing devices for biomedical applications.

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3.2 Dye release study
Dissolving microneedles have different mechanism of releasing drug than other types where they
use paddle over disk method [23]. However dissolving microneedles involves mechanism of

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drug release upon dissolution of needles [24]. In the present investigation trypan blue (used as
the model drug) release rate was studied to illustrate the effective delivery through dissolving
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collagen microneedles. Studies have been demonstrated for drug release profile using PBS
(Phosphate Buffer) as release medium [25,26] with the intention of mimicking the pH condition
as human skin. However, for the present investigation we have used 0.8% NaCl to maintain the
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pH as well as considering the salinity of the skin and comparison was made while using distilled
water as release medium. The results demonstrated that the collagen microneedles can readily
dissolve and release the material into the medium whereas the dissolution rate in distilled water
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was faster than that of NaCl solution (Figure 4). Ten microneedle patches of trypan blue loaded
collagen-PVP were considered for the study using distilled water as release medium and average
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rate of release of trypan blue from the collagen microneedles and the average loading capacity
were determined (Figure 4, 5). It is obvious from the results that loading capacity and the release
rate of trypan blue relies on the size of the needle, more heighted the needle - longer the time
release as it can accommodate more amount of dye than smaller ones.
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3.3 Histological studies

While defining the specifications for microneedles, the basic requirement of its skin
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penetration potential must be defined [21]. Here, mice skin had been experimented to check for
insertion of different sized microneedles whereas figure 6 is demonstrated with 400µm
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microneedle potential. The microneedle is seen to be successfully penetrated to the skin. Hence it
can be said that our formulation is strong enough to perform the appropriate action which
predicts their collagen or drug delivery capabilities.
4. Conclusion
The present investigation demonstrated the synthesis of dissolving collagen microneedles
using micro-molding technique optimizing various process parameters such as W/V ratio of
biopolymers, microneedle height, pressure drop, and duration of solidification play significant
roles to generate microneedle. The correlations and dependence of each parameter was analyzed
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by Taguchi L9 and revealed the most suitable conditions for effectual microneedles. According to
the values of signal to noise ratio, it is evident that the height of the microneedle and time are the
major contributors. Histological imaging clearly proves the penetrating ability of the
microneedles and addition of trypan blur dye to the microneedle to analyze the rate of release
supports the concept of collagen and drug delivery through microneedles. The work presented
here is evidence of possible microneedle synthesis containing collagen as the main biopolymer

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which is the most versatile scheme to our knowledge. It offers the tunable platform as the easiest
processing method with gentle fabricating conditions and proposes a promising collagen
administrating device.

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Acknowledgement:

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This work was funded by SKU-UTEP MS Program: 2011-0216 and supported by School of
Pharmacy, The University of Texas at El Paso.

Conflicts of interest
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The authors have no financial interests.
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Figure Captions

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Figure 1 Method of preparing collagen – PVP microneedles with trypan blue dye. (a) The
microneedle mold filled with a mixture of 60µl of trypan blue dye (0.4%) and 60µl collagen –
PVP solution; (b) Filled micromolds placed in the vacuum oven; (c) Removal of excess solution

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leaving behind solutions inside the micro-slots to form the microneedles; (d) Adding collagen -
PVP solution to from the bed of the microneedle patch.
Figure 2 A) 300µm microneedle patch B) 400µm microneedle patch C) 500µm microneedle

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patch prepared by micromolding of collagen-PVP solution as per procedure demonstrated in
Figure 1.

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Figure 3 Plot for S/N ratio for each control factor derived from table 3, exhibiting that the
microneedle height has the most statistical significance on the microneedle dimension.
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Figure 4 Trypan blue release in distilled water and 0.8% NaCl from each size i.e. 300, 400 and
500 µm of microneedles. The bars represent the standard deviation of experimental results
performed in triplicates.
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Figure 5 Loading profile of trypan blue in microneedle. Demonstration of trypan blue holding
potential of microneedles of different sizes. The bars represent the standard deviation of
experimental results performed in triplicates.
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Figure 6 Histological image of 400µm collagen microneedle inserted into the mouse skin and
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stained with eosin.

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Table I: Taguchi L9 orthogonal array. The height, pressure drop, duration and the concentration
are the four control factors respectively. The 300µm (1), 400µm (2) and 500µm (3) are the three
levels for the height being the first control factor. The second control factor is the pressure drop
in which -0.02MPa (1), -0.03Mpa (2) and -0.04MPa (3) are the three levels. The duration of the
microneedles in the vacuum oven is the third control factor where 12Hr (1), 18hr (2) and 24Hr

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(3) are the three levels of the third control parameter. The fourth control factor is the
Concentration of Collagen & PVP in distilled Water where the three levels are 8% wt./vol (1),
10% wt./vol (2)and 12% wt./vol (3).

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Experiment Control factor 1 Control factor 2 Control factor 3 Control factor 4

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No. (Height) (Pressure Drop) (Duration-Oven) (Concentration)

1 1 1 1 1

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2 1 2 2 2
3 1 3 3 3
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4 2 1 2 3
5 2 2 3 1
6 2 3 1 2
7 3 1 3 2
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8 3 2 1 3
9 3 3 2 1
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Table II: Taguchi L9 setup of experiment with modulus of deviation of volume ratios and signal
to noise ratios

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Collagen-PVP

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Concentration Modulus of Signal
No. of Microneedle Pressure Time in distilled deviation of volume to noise
Experiments Height (µm) Drop (Hours) Water (% ratios ratios

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(MPa) wt./vol.) Trial Trial Trial (SN)
1 2 3
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1 300 -0.02 12 8 0.007 0.065 0.011 31.27
2 300 -0.03 18 10 0.110 0.173 0.021 19.88
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3 300 -0.04 24 12 0.090 0.180 0.098 18.23

4 400 -0.02 18 12 0.074 0.260 0.227 14.56
5 400 -0.03 24 8 0.190 0.087 0.207 15.85
6 400 -0.04 12 10 0.070 0.064 0.067 23.48
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7 500 -0.02 24 10 0.201 0.182 0.336 12.42

8 500 -0.03 12 12 0.231 0.138 0.156 15.14
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9 500 -0.04 18 8 0.069 0.320 0.003 17.68

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Table III: Response table for S/N ratios

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Level Height Pressure drop Time Concentration

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1 21.54 18.08 22.25 19.41

2 17.56 16.27 15.59 18.01
3 13.82 18.57 15.08 15.50
Delta 7.72 2.30 7.17 3.91
Rank 1 4 2 3
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Table IV: Analysis of variance

Control Factor Degree of Sum Squared Mean Square Percentage F ratio

Freedom (DOF) (SS) Contribution
Needle Height 2 99.73 49.86 38.24% 7.00
Pressure Drop 2 14.24 7.12 5.46% -
Concentration 2 47.45 23.73 18.20% 3.33

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Time 2 99.37 49.69 38.10% 6.98
Error and Noise 0 0 0 0 -

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