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. Author manuscript; available in PMC: 2022 Jan 5.
Published in final edited form as: Int J Pharm. 2020 Nov 5;592:120050. doi: 10.1016/j.ijpharm.2020.120050

A crossover clinical study to evaluate pain intensity from microneedle insertion in different parts of the oral cavity

Stephany Di Carla Santos 1, Nádia Cristina Favaro Moreira 1, Henrique Ballassin Abdalla 1,2, Gabriela Gama Xavier Augusto 1, Yuri Martins Costa 1, Maria Cristina Volpato 1, Francisco Carlos Groppo 1, Harvinder Singh Gill 3,*, Michelle Franz-Montan 1,**
PMCID: PMC7856577  NIHMSID: NIHMS1649086  PMID: 33161036

Abstract

The objective of the present study was to evaluate discomfort and safety of microneedle (MN) insertion in several intraoral regions. A device was developed to standardize MN insertions. MNs were inserted in the following regions of the oral cavity: gingiva, palatine alveolar process, buccal mucosa, dorsum of the tongue and inner portion of the lower lip. Perforations from MNs post insertion were confirmed with topical gentian violet stain. Pain was evaluated in a randomized, double-blinded, crossover study in 30 volunteers. Each volunteer received a MN patch, a 30G hypodermic needle (positive control) and an identical MN patch with its needles laying flat in the plane of the patch (negative control). Adverse events were visually evaluated immediately after (0 h) and 24 h post MN application. The application device developed a consistent application force (10 N) and promoted perforation of all individual MNs on a patch. At all sites, insertion of the hypodermic needle promoted more pain when compared to the negative control (p <0.001). Application of the MNs promoted less pain than the hypodermic needle (p<0.05), but slightly more pain as compared to the negative control (p <0.05) at all sites except the tongue, where the MN did not differ from the negative control (p> 0.05). Hypodermic needle caused bleeding at all insertion sites. In contrast, MNs did not cause bleeding at most sites except in some cases of insertion into the hard gingiva and the palatine alveolar process where tiny blood spots appeared immediately after MN application for few of the MNs on the patch. There were no cases of bleeding at 24 h post MN application. In conclusion, MNs can perforate different sites of the oral cavity in a safe and significantly less painful manner as compared to the 30G hypodermic needle. Thus, analogous to the skin, MN-based approaches could be an attractive approach for drug delivery in the oral cavity.

Keywords: Clinical trial, Drug delivery, Microneedles, Oral mucosa, Topical administration, Transmucosal

Graphical Abstract

graphic file with name nihms-1649086-f0001.jpg

1. INTRODUCTION

Topical administration of drugs in the oral cavity continues to be challenging. This is because the mucosal epithelial layer that lines the oral cavity offers a considerable barrier to drug diffusion, and it is difficult to maintain the formulation at the site of application due to the continuous washing action of saliva and movements in the oral cavity occurring from speaking, chewing and swallowing (Hassan et al., 2010; Hearnden et al., 2012; Kulkarni et al., 2009). Consequently, to obtain local effects in the oral cavity, local injections in the oral cavity are often made, which are painful and cause discomfort to patients.

Recently, a new approach of drug delivery that uses microneedles (MNs) has been used for drug and vaccine delivery into the oral cavity. For example, a study demonstrated in vivo the feasibility of delivering vaccines through the lip and tongue of rabbits (Ma et al., 2014), while another demonstrated efficacy of vaccine delivery through the buccal region in mice using MNs (Wang et al., 2015). While MNs have only recently received attention for intraoral drug delivery, in contrast, they have been studied for over two decades for the purpose of drug and vaccine delivery into the skin (Jin et al., 2018; Prausnitz, 2017). Indeed, a broad range of molecules including small molecules, peptides, proteins, oligonucleotides, viruses and even particles have been delivered into the skin using MNs (Ingrole and Gill, 2019). Based on the success of MNs for transdermal delivery and the recent favorable results from their use in the oral cavity, it is clear that MNs have the potential to make a significant contribution towards oral cavity transmucosal delivery of several drugs of both small and large molecular weights including protein-based biologics that can produce diverse local or systemic effects such as antitumoral, antimicrobial, anesthetic, and antiinflamatory effects.

One of the salient features that has led to the popularity of MNs is the fact that they are minimally invasive and painless (Gill et al., 2008). The use of MNs is also associated with several other advantages such as the possibility of self administration by patients and promoting good tolerance without inducing significant edema or erythema (Bal et al., 2008; Gill et al., 2008; Haq et al., 2009). While the painless nature of MNs for skin administration has been established in humans (Gill et al., 2008; Gupta et al., 2011a; Wermeling et al., 2008) this aspect has not yet been established in the context of the oral cavity. Before MNs can be more extensively employed for oral cavity transmucosal drug delivery, their painless nature and associated adverse effects from insertion in human oral cavity must be better understood. Accordingly, in this paper, we present the first clinical trial reporting the safety and painless characteristic of MN application in different sites of the oral cavity. We applied MN patches in different regions of the oral cavity using a simple applicator to achieve consistent application force. We demonstrate here that MNs are significantly less painful than insertion of a hypodermic needle into the oral cavity.

2. MATERIAL AND METHODS

2.1. Clinical study design and treatments

Pain and safety of MN insertion into oral cavity was accessed in a randomized, crossover, double-blinded, placebo-controlled clinical trial including 30 male volunteers. For each volunteer, treatments were performed at five different locations of their oral cavity, namely the lip, buccal surface, tongue, palate and gingiva. At each of these locations three different treatments were applied: a MN patch whose needles were sticking out perpendicular to the base surface (MN group), a MN patch whose needles remained flat and in plane with its base as a negative control (FL group), and a 30G hypodermic needle (NE group)

The hypodermic needle was inserted 3 mm deep in lip, buccal surface and tongue, and up to periosteum contact in the palate and gingiva. A 3-mm insertion depth has been selected for insertion of a hypodermic needle into the lip, buccal surface and tongue to simulate injection of drug into local tissues. This is the shortest needle length that can penetrate the tissues for injection without backflow of the injected liquid. Periosteum contact was selected in the case of palate and gingiva because 3 mm length is too long for injection into the palate and gingiva.

Each volunteer received a total of 15 treatments. The order of the intraoral application site (lip, buccal, tongue, hard palate, or attached gingiva), the treatment order (MN, NE or FL) and the side of application (right lateral or left lateral) were all independently randomized. Each site received a total of all three treaments, according to the example in Table 1.

Table 1.

Treatment randomization for volunteer 1 wherein sequence of regions, sequence of treatments and application sides (left or right) were all independently randomized.

Volunteer 1 Sequence of regions Sequence of treatment Application Side
1) Palate MN Left
FL Right
NE Right
2) Buccal FL Right
MN Left
NE Left
3) Gingiva NE Left
FL Right
MN Left
4) Lip FL Right
NE Right
MN Left
5) Tongue MN Right
NE Left
FL Right
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The volunteers were blinded to the treatments. Pain associated with each treatment was evaluated by a different blinded dentist using a Visual Analogue Scale (VAS) consisting of a 10-cm line without demarcations, containing only the terms “no pain” at the far left, and “unbearable pain” at the far right (Hjermstad et al., 2011).

All insertions were performed by the same trained dentist with a gap of 30 seconds between each insertion. The volunteers received all the treatments in the following regions of the oral cavity: lip (inner lower), buccal (cheek: 1 cm behind the mouth angle), tongue (dorsal surface), hard palate (alveolar process, anterior region), and attached gingiva (between central and lateral upper incisors) as shown in Figure S1. To facilitate insertion of MNs and the hypodermic needle into tissues of the lip, the buccal and the tongue that are flexible and have no rigid backing of a bone, a wooden spatula was placed underneath the tissues prior to the treatments.

Adverse events associated with MN application were evaluated immediately after the treatment (0 h) and 24 h later. Bleeding, ulceration, bruising, redness and swelling were evaluated as part of the adverse event assessment. In addition to visual inspection, sites were photographed. The site to be photographed was randomized and the blinded dentist was unaware of the site to be photographed ahead of time to prevent any bias of insertion. Moreover, to examine whether the MNs were getting damaged or bent after application, all the used patches were evaluated by scanning electron microscopy (SEM) (JEOL JSM 5600 PV, coupled with EDX System). Samples were observed at 15 kV from both the front and the side.

Figure 1 shows an overview of the clinical study design.

Figure 1.

Figure 1.

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Experimental design of the clinical study to evaluate the discomfort and adverse events resulting from the application of MNs in the oral cavity of human volunteers.

2.2. Ethics

The Brazilian National Committee for Ethics in Research /National Health Council/ /Ministry of Health) (CONEP/CNS/MS) approved this research (CAAE: 39194814.8.0000.5418) and has been registered on the ClinicalTrials.gov website (Identifier Number: NCT03855397). The selected volunteers were submitted to a clinical oral cavity inspection and they signed a written informed consent according to Resolution 466/12 from the Brazilian National Health Council. Inclusion criteria was healthy men with good oral health. The following exclusion criteria were adopted: smokers, alcoholics and intake of medication that would alter pain perception or local anesthesia in the oral cavity in the last 2 weeks prior to the study.

2.3. MN patches

MN patches were prepared based on a previously described method (Ma et al., 2014; Shakya et al., 2019). Briefly, a MN patch comprising 55 MNs each 750-µm long and 200-µm wide was drafted in AutoCAD software (Autodesk, Cupertino, CA, USA) and then was cut into 50 µm thick stainless steel sheets (stainless steel 316) using a wet etching technique (Tech-Etch, Plymouth, MA, USA). The MN patches were next washed under running water and dried using compressed air. Following this process, each MN of the patch was manually bent 90° “out of plane” under the microscope. Each MN was found to be about 700 µm long in their bent state.

2.4. Application system

An applicator was developed to apply the MN patch in the oral cavity. The applicator comprised a 5-mL syringe (Descarpack®, lot: SLLAA0014) in whose bore a spring with an outer diameter of 10 mm and a force constant of 2.55 N/mm was placed. By pressing the plunger of the syringe by a fixed distance, the spring deformation was controlled, which in turn helped to control the application force. A MN patch (either with MNs bent at 90 ° or with MNs still flat) was fixed on the flat face of the plunger and the complete system was sterilized in an autoclave (121°C, 1 atm, 15 minutes) prior to application into the oral cavity.

2.4.1. Characterization of MN applicator

The force induced by spring compression in the applicator was measured by manually pressing the syringe plunger through a distance of 2 mm with the help of markings on the syringe barrel (the distance between each marking was 2 mm). Ten independent applicators were tested by placing each against the surface of an analytical weighing scale and pressing it down in order to measure the force (kg) generated (10 x 10 =100 measurements). The measurements were repeated on two different days, resulting in a total of 200 readings.

2.4.2. Confirmation of oral mucosa perforation following MN application

Insertion of MNs in a given patch into the oral mucosa was verified by visually confirming microperforations produced from the individual MNs on the patch. Briefly, after application of the MN patch at different sites in the oral cavity using the applicator device, the areas were stained with 1% gentian violet to stain the microperforations created by the individual MNs on the patch. The stained regions were photographed using a digital camera (Canon EOS XTI; macro 100 Canon; flash canon ring mr14 eXII).

2.5. Statistical analysis

The pain intensity was reported as median and interquartile range (IQR), unless otherwise noted. Normal distribution was assessed with the aid of Kolmogorov-Smirnov test and log10 transformations were applied for the pain intensity values, considering an alpha level of 5% (p < 0.05).

Repeated-measures two-way ANOVA was computed to assess differences in the pain intensity (log10 tranformed values). The following within-subject factors were considered: site (five levels – palate, tongue, lower lip, buccal mucosa and gingiva) and type of treatment (three levels – MN, FL and NE). Where appropriate, post hoc analyses were performed using Tukey Honestly Statistical Difference (HSD). The significance level was set at 5% (p = 0.05).

Cochran’s Q test was applied to compare the proportions of pain report, i.e., VAS > 0, among the sites following the MN application. Where appropriate, post hoc analyses were performed using Dunn’s test. Finally, McNemar’s test was applied to compare the proportions of bleeding between the MN and NE treatment. The significance level was set at 5% (p = 0.05).

3. RESULTS

3.1. Characterization of MNs and the applicator device

Figure 2A shows a photograph of a representative patch used in the present study. The patch is shown placed on a human index finger to provide contextual information regarding the size of the MNs and the patch. Each MN patch was circular in shape with a diameter of 15 mm and contained 55 MNs each about 700 µm long in their bent state. Figures 2B and 2C are SEM images of an unused patch showing the side and front views, respectively. Figure 2C also shows a 30G hypodermic needle, which is representative of the positive control that was used in this study.

Figure 2.

Figure 2.

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(A) A MN patch containing 55 MNs each extending about 700 µm in length from the basal surface. (B) SEM image of a MN patch before use showing its side view, (C) and SEM image of a MN patch before use showing its front view together with a 30G hypodermic needle.

We developed an applicator device as shown in Figure 3A, to apply the MN and FL patches in different parts of the oral cavity with a consistent force. The top and the bottom images in Figure 3A show the applicator-state before and after pushing on the syringe plunger. The two consecutive divisions marked on the barrel of the 5 mL syringe correspond to a 2 mm separation (Figure 3A) and were used as visual guides to press the spring by 2 mm. The MN or FL patch was attached on the flat end of the syringe plunger as depicted in Figure 3B. To evaluate the consistency of force generated by pushing on the plunger, we performed an experiment in which the plunger (without MNs attached) was manually pushed vertically-down on an analytical weighing scale. Figure 3C shows the variation of the force values obtained when different devices were tested on two consecutive days (n=200). By producing a 2-mm spring deformation, the applicator developed a force of approximately 1 kg (~ 10 N), with a variation coefficient of about 7%. The measured force was higher than the expected force of 5.1 N computed from the spring force constant (2.55 N/mm x 2 mm). This can be attributed to friction between the plunger and the barrel, which increases the force required to compress the spring.

Figure 3.

Figure 3.

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(A) Photographs of the MN applicator device before (top) and after (bottom) pushing on the plunger and spring. The vertical dotted lines depict the start and stop positions of the plunger. (B) Photograph showing a MN patch attached on the syringe plunger surface. (C) Force (N) measured by pushing the applicator on an analytical weighing scale on two different days (10 devices each pressed 10 times on two separate days, n = 200).

3.2. Confirmation of MN penetration into different tissues.

To verfity that MNs can penetrate into different tissues of the oral cavity sites, we applied gentian violet on the tissue after application of MNs. Gentian violet is dark blue/violet in color and can penetrate the micropores from MN insertions, and can thus help to visually identify insertion points of individual MNs on a patch. Figures 4 A and 4B illustrate dot patterns in the non-keratinized tissues (inner portion of the lower lip and buccal tissue, respectively), and Figures 4C and 4D illustrate dot patterns in the keratinized sites (palate and gingiva, respectively). The blue dots in the figures correspond to MN insertion points and confirm the insertion of MNs in different regions of the oral cavity. In the case of the tongue, it was hard to stain MN perforation sites because the taste buds also got stained with gentian violet (Figure S2)

Figure 4.

Figure 4.

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Images of keratinized and non-keratinized sites immediately after MN insertion and 1% gentian violet application: (A) inner portion of the lower lip; (B) buccal; (C) palate; (D) and gingiva.

3.3. Pain after MN patch application in the oral cavity

Figure 5 shows the distribution of pain intensity following different treatments in different sites of the oral cavity. Overall, insertion of the 30G hypodermic needle (NE) evoked the highest pain intensity regardless of the application site (p < 0.001). Furthermore, for all sites, application of MNs evoked lower pain intensity when compared to the positive control (NE), which means that, overall, MNs were less painful than the 30G hypodermic needle (p < 0.05). The pain intensity following MN application was higher than the negative control (FL) (p < 0.05) for all the application sites except the tongue, where the MN and FL treatments presented similar pain intensity values (p > 0.05). The number of volunteers who reported MNs to be more painful than the negative control (FL group) were 3/30, 13/30, 1/30, 1/30 and 4/30 for the palate, lips, tongue, gingiva and buccal sites, respectively.

Figure 5.

Figure 5.

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Median (interquartile deviation) of pain perception assessed through VAS (in mm) due to the application of the microneedle patch (MN), negative control (FL) and positive control 30G needle (NE) for the 5 sites of the oral cavity: (A) palate; (B) lip; (C) tongue; (D) gingiva; (E) buccal. Repeated-measures two-way ANOVA, followed by the Tukey test. * p <0.05; *** p <0.001 (n = 30).

3.4. Effect of application site on pain

To determine whether application sites influence pain, we compared i) the VAS values at different sites, and ii) the percent of volunteers who reported pain at the different sites for the NE and MN groups separately.

In the case of NE groups, no significant difference was observed in VAS values from NE insertion at the different sites (Tukey: p > 0.050). The percent volunteers who reported pain (i.e., VAS > 0) from insertion of NE in palate, lip, tongue, and gingiva was 97% while 90% reported pain in buccal area. No significant difference in percent volunteers reporting pain was observed (Cochran’s Q test: X2 = 2.66 and p > 0.050).

In the case of MN insertion, no significant difference was observed in VAS values from MN insertion at the different sites (Tukey: p > 0.050). The percentage of volunteers who reported pain (i.e., VAS > 0) in the MN group were, 57%, 60%, 60%, 77%, and 83% in buccal, palate, tongue, gingiva, and lip, respectively (Cochran’s Q test: X2 = 14.11 and p = 0.007). Post-hoc Dunn’s test indicated that the percentage of volunteers who reported pain following MN treatment was higher in the lower lip (83%) as compared to buccal mucosa (57%) (p = 0.029). For the other sites, the differences were not significant (p>0.050).

3.5. Incidence of adverse effects after MN application in the oral cavity.

Table 2 describes the incidence of bleeding due to the application of the treatments in the different sites of the oral cavity immediately after the treatments (0 h). As expected, no bleeding was observed after FL use. From MN insertion, no bleeding was observed in the lip, tongue and the buccal region, however in 10% and 13.3 % of the volunteers, few tiny spots of blood (maximum 3 spots per patch) were seen in the palate and the gingiva. Since each MN patch has 55 MNs, this means that at most 5% of the MNs led to blood spots. The spots were so small that they remained as stationary droplets. In contrast, when 30G needle was applied, bleeding was observed in most volunteers at all sites. Overall, the proportion of volunteers who presented bleeding following the postitive control NE (83%) was higher than MN (20%), regardless of the site (p < 0.001).

Table 2.

Incidence of bleeding after the application of the different treatments (MN: microneedles; FL: negative control; NE: positive control) in the different evaluated sites.

MN (n=30) NE (n=30)
Palate 3 (10%) 11 (36.7%)
Lip 0 (0%) 2 (6.7%)
Tongue 0 (0%) 16 (53.3%)
Gingiva 4 (13.3%) 13 (43.3%)
Buccal 0 (0%) 4 (13.3%)
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Bleeding stopped within few minutes and it was no longer observed after 24 h. No cases of redness, swelling, bruising and ulceration were observed after the use of the treatments either immediately or after 24 h (MN, NE, and FL).

Figure 6 shows photomicrographs of MN patches after application at all the application sites. MNs remained upright after insertion into the 3 different types of mucosa of the oral cavity (keratinized, non-keratinized and specialized). In addition, these photomicrographs also reveal the mechanical robustness of the MNs after use, confirming that there was no fracture of MNs inside the tissues.

Figure 6.

Figure 6.

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Photomicrographs obtained by scanning electron microscopy (SEM) of used patches after use in all the application sites: lip (A and B), buccal (C and D), tongue (E and F), palate (G and H) and gingiva (I and J). Images on the left show front view and, on the right, show side view.

4. DISCUSSION

The present study demonstrates for the first time the painless nature of MNs upon MN application in different regions of the oral cavity of humans. This study is relevant because MNs at different regions of the oral cavity could be an important tool for local drug delivery. However, there are no studies that support the painless characteristic of MNs in the oral cavity. Therefore, in this study our objective was to evaluate pain after insertion of MN arrays in different regions of the oral cavity. We selected different locations of the oral cavity for evaluation because they could be used for MN-based drug and vaccine delivery. For example, MNs could be applied on the gingiva to deliver antibiotics and antispectic agents that are often applied topically to treat periodontitis (Schwach-Abdellaoui et al. 2000). In the case of lips and tongue, MNs could help to improve symptom relief of oral mucosal pain from burning mouth syndrome (McMillan et al. 2016). The use of MNs as an alternative delivery device at the palate is also an attractive option for improving effectiveness of topical anesthesia during local surgical procedures involving the palate (Franz-Montan et al. 2017). And finally, the buccal mucosa has caught attention in topical administration for systemic effects (Senel and Hincal 2001, Şenel et al. 2012). In addition, this site has also been reported for vaccine delivery (Kraan et al. 2014). Interestingly, some studies have already demonstrated the feasibility of MNs in inducing oral mucosal and systemic immune response after MN application at buccal and lip mucosa (Ma et al. 2014).

We opted to evaluate a patch with MNs of about 700 µm in length. This selection was based on two criteria. First, the epithelial tissue, which is the main barrier against penetration of drugs presents a wide thickness variation (about 70 µm in the gingiva, and over 500 µm in the buccal) (Alves et al., 2018; Squier, 2011); therefore, we decided to evaluate a MN longer than 500 µm. Second, a previous study has examined the effect of MN geometry on pain from insertion in the skin of human volunteers (Gill et al., 2008). In that study it was shown that MNs about 700 µm in length are significantly less painful as compared to a hypodermic needle, but as the MN length was increased beyond 700 µm to about 1000 µm, the pain intensity increased and no volunteers reported MNs to be completely painless. Accordingly, we selected a MN length of about 700 µm for evaluation in the oral cavity. It should be noted that the positive control hypodermic needle used in the present study is a smaller gauge needle (30G) unlike the skin study, which used a 26G needle. We used the 30G needle because it is the most common needle gauge used for infiltrative injections into the oral cavity (Reed et al., 2012).

In this study we confirmed that the application of MNs in different regions of the oral mucosa promotes lower pain intensity as compared to a 30G hypodermic needle inserted 3 mm deep in the lip, buccal and tongue or until periosteum contact in the gingiva and palate regions. These results corroborate with results from previous clinical studies performed on the forearm skin of healthy volunteers, in which MNs of different geometries promoted less pain perception when compared to hypodermic needle insertion (Gill et al., 2008; Gupta et al., 2011b), and by Haq et al., who demonstrated that the MN application induced lower pain perception in the skin of the buttocks of volunteers when compared to a hypodermic needle (Haq et al., 2009).

A weak correlation was found between pain sensation and the different application sites. The number of volunteers who reported pain (i.e, VAS > 0) increased from buccal, palate, tongue, gingiva, to lip region. The difference was significant only between buccal and lip region, indicating that while use of MNs in all areas of the mouth was well tolerated, the areas that may be preferred are the buccal region. Interestingly, even the palate and the gingiva, which are supported by a harder tissue were well tolerated for MN insertion. The lip was found to be the most sensitive region of the oral cavity.

When pain from MNs was compared to pain from the negative control (FL group) it was found that MNs generated a higher pain perception in all oral cavity sites except the tongue. This observation is similar to the trend reported in human forearm skin (Gill et al., 2008), where again the MNs induced more pain than a negative control. It should however be noted that the number of subjects who reported MNs were more painful than the negative control were under 13% at all sites except the lips where 43% of volunteers reported more pain than the negative control.

According to the gate control theory of pain, sensory inputs from low threshold tactile fibers can inhibit spinal nociceptive neurons (Melzack and Wall, 1965). This relationship between large and small fiber afferents has also been demonstrated within the trigeminal innervation (Costa et al., 2017; de Moraes Maia et al 2014) and could explain the lower pain intensity reported for the tongue. Moreover, the tip of the tongue is densely innervated with mechanoreceptores (Komiyama and De Laat, 2005). Therefore, it can be argued that the concomitant mechanical non-painful stimulation of the tongue that was necessary to appropriately apply the MN (stretching the tongue and placing it on a wooden spatula to prepare for MN insertion) may have decreased the nociceptive signalling.

Another novelty of this study is that it introduces for the first time an applicator device that allowed controlled MN insertions in different parts of the oral cavity, including the difficult to access areas such as the palate. Controlling the force of MN application is important because it helps to reduce bias introduced by poorly controlled insertions. To the best of our knowledge, there is no study reported in the literature, which provides force of insertion required for MN insertions into the oral cavity tissues. In contrast, a range of insertion forces for MN insertions into the skin have been reported, which span few Newtons (Davis et al., 2004) to tens of Newtons (Cheung et al., 2014). The applicator design we created is simple and inexpensive. We tested the applicator in a preliminary study in humans and found that about 10 N force was sufficient to allow MN penetration into the different mucosal regions. Lee and coworkers (2017) also describe that they used a force of 12 N for the application of dissolvable MNs made of carboxymethylcellulose and gelatin into the skin, however, these authors did not detail how this force was predetermined and standardized (Lee et al., 2017). Other authors have developed an applicator device for a MN patch containing 36 solid platinum-coated silicon MNs of 180 and 280 µm in lengh using a 2-mL syringe similar to the present study (Haq et al., 2009).

However, these authors did not control the force of insertion, and the syringe plunger tip was simply used to ‘hold’ the MN patch. In contrast, we have introduced a spring in the syringe barrel to control the force of insertion. This simple applicator could be modified further to increase the force-control.

After insertion, MNs remained intact and showed no signs of bending or fracture (as confirmed by SEM photomicrographs). MNs were found to be safe for use in different sites of the oral cavity. No side effects such as ulceration, bruising, redness or swelling, were observed from the MNs or the applicator device. In some subjects, tiny blood droplets were observed in the regions of hard palate and gingiva after MN use. This could be explained by the presence of a thin overlying epithelium in these locations, which can lead to a higher probability of MNs reaching the blood vessels (Squier, 2011). Bleeding incidence was significantly higher in all locations when the hypodermic needle was used and a visibly larger volume of blood was generated from hypodermic needles as compared to MNs. Similar results were reported by Ma et al. (2014), who reported a greater index of bleeding in the oral mucosa of the rabbits (internal region of the lip and tongue) after the use of a hypodermic needle when compared to MNs (Ma et al., 2014).

In conclusion, the 700 µm long stainless steel solid MN patch applied with the aid of a device that standardizes the application force was demonstrated to be a safe and significantly less painful as compared to a hypodermic needle. Based on these results, we propose that all evaluated sites (lip, buccal, tongue, gingiva and palate) in the oral cavity are promising sites for drug delivery using MNs, which could offer a promising way to improve the clinical efficacy of intraoral topical formulations where local and/or systemic effects are desired.

Supplementary Material

1

ACKNOWLEDGEMENTS

Financial support was provided by the São Paulo Research Foundation (FAPESP, grants #2012/06974-4 and #2015/50004-8). S.C.S. received a masters’ scholarship from FAPESP (#2016/24057-0). HSG was partially supported by the National Institutes of Health (NIH) [grant numbers R01AI121322 and R01AI135197] and in part by funding from Texas Tech University.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

CONFLICT OF INTEREST STATEMENT: HSG is a co-inventor on a patent related to coated microneedles. This potential conflict of interest has been disclosed and is managed by Texas Tech University.

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