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Describe about the Physical Performance of Different Dissolving Nanopatch Formulations?

Nanopatch method of vaccination has been implemented now a day to provide a replacement over the standard methods, which includes delivery of the vaccine to the patient with the help of a needle. Nanopatch technology provides a cost-effective, needle-free and more precise way of delivering vaccine to evoke immunological responses. Nanopatches resembles a small chip with gold coated silicon wafers lined by numerous microneedles that penetrate the skin to administer the vaccine.

Nanopatch formulation helps in the manufacture of proper nanopatches that are widely used for medical purpose. Formulation of nanopatches includes various fabrication techniques. Primarily nanopatches are fabricated with different sugar molecules like sucrose, trehalose, sorbitol and mannitol followed by FT-FIR analysis.The useful role of the various sugar molecules helped in determining the qualitative properties for a better understanding of the nanopatches properties, and the FT-FIR data assisted in identifying the amorphous and the crystalline nature of the nanopatches formulated. Rhodamine-dextran fluorescence method also helped in knowing about the morphological and physical changes from that of pure CMC dNPs.

Furthermore, nanoindentation assisted in revealing information regarding the bulk properties of the various CMC sugar/polyol dNPs. The SEM images thus obtained helped in providing data regarding the surface property of the CMC/sugar polyol dNPs indicated by the presence of buckle structure.

Keywords: nanopatches, vaccination, formulation, FT-FIR, SEM

Vaccination refers to the administration of an antigenic material in the form of a vaccine to stimulate the immune system of an individual for developing immunity against a particular pathogen or disease. Vaccination plays a vital role in preventing the spread of infectious diseases (Josefsberg and Buckland, 2012). The method of prevention includes administration of the vaccine, which primarily comprises of the active agent (inactive or attenuated form) of the antigens into individuals to develop an immunological response to a particular disease (Schild et al. 2015). The method of vaccination includes administration of the vaccine, which primarily consists of an efficient and safe injection of the vaccine (Knight-Jones et al. 2014).

Apart from the traditional method of vaccination, in recent days, a new method of immunization has been implemented which is termed as Nanopatch (Fernando et al. 2012). Nanopatch aims to provide an efficient, optimized and differentiated needle-free vaccine delivery system, which tends to be safe and cost effective. Nanopatch technology refers to a pain-free vaccine delivery system to develop a protective immunological response using a dose, which is one-hundredth of the dose as required by the conventional mode of administration using a needle or syringe (Corrie, Depelsenaire and Kendall, 2012). Nanopatches are like small chips that are primarily made up of silicon (gold coated) wafers lined with infinite numbers of microneedles (Ravi, Sadhna and Chawla, 2015). The micro-needles are coated with specific vaccine that penetrates the top layer of the skin where special types of immune cells help the body to respond to a particular infection (McCaffrey, Donnelly and Mc Carthy, 2015).

The given article helps us in understanding the physical performance of the different kinds of nanopatch formulations. The various formulations associated helps in the manufacture of moulds by suitable methods, which include dissolving of nanopatches (Fernando et al. 2012). The role of various types of sugar molecules related to the particular method has also been highlighted in the given article. Investigation of the crystalline structure of the nanopatches by applications of the particular biophysical technique also helps in developing knowledge regarding the structural and functional orientation of the respective nanopatches (Corrie, Depelsenaire and Kendall, 2012). Thus, the present article emphasizes on exploration and interpretation of the results associated with careful experimentation for developing knowledge and data regarding nanopatches formulation.  

Biomedical Engineering

Formulation of dissolving Nanopatches (dNPs)

Fabrication of the nanopatches was initially done by the casting of the moulds using silicon nanopatch masters, placed into polydimethylsilicone (PDMS) and was then allowed to dry. Nanopatches were removed, and moulds were carefully placed in a 24 well plate with the addition of 50 µl of a sugar-polyol formulation (Grande et al. 2013).

The sugar molecules used as a component of the formulation included Sucrose, Trehalose, Sorbitol and Mannitol, which was mixed and centrifuged at 3000 g for 1 hour at 25ºC for delivering the sugar molecules into the projection moulds. The formulation was then left to dry.

After proper drying up of the formulation, 50 µl of 2.1 mM carboxymethylcellulose (CMC) was added to the moulds. The moulds containing the dry beads were then placed in a sealed desiccator at 22 ºC under nitrogen thereby allowing the dNPs to dry up within 4-6 hours.  Finally, the dNP array was stored in a sealed desiccator under nitrogen at 22 ºC.  The arrays were safely removed and were then analyzed using a stereo microscope. The table given below outlines the weight-to-weight ratio of CMC to sugar/polyol formulation in association with the given study


1 : 1.25

1 : 1.7

1 : 2.5

1 : 3.3

1 : 3.75

1 : 5

1 : 6.25

1 : 6.7

1 : 7.5

1 : 8.3

1 : 10

1 : 12.5

1 : 15

1 : 20

1 : 25

1 : 30


Table 1: Comparative w/w CC: sucrose, trehalose, sorbitol or mannitol

The method was applied for measuring deflection in projection by applying force on individual projection (mainly at the top). Thus, a cylindrical probe (40µm diameter) was applied in axial direction onto the dNP projections. 10µl of sugar/polyol formulation were mixed with the moulds for studying the fluorescence microscopy (Lua et al. 2014). Rhodamine-dextran (M/W 3000 g) was added to the solution with a final concentration of 0.1mg/mL. To compare the delivery of the dNPs, C14 radiolabelled ovalbumin protein was used to trace the dNP formulations. C14 ovalbumin solution was prepared in such a way that each mould received 10 µl of the solution with a minimum of 25 nCi14 C ovalbumin.

Fourier transform infrared (FT-FIR) spectroscopy helps in detection of the microcrystalline nature of the associated dNP samples. The use of synchtron radiation helps in analyzing the sugar/CMC formulations at a much higher resolution (Kunz, 2013) Sample preparation was done by pipetting 100 µl of each sample into three copies on the surface of a glass slide wrapped with parafilm. The formulations were then allowed to dry overnight in a desicattor, thereby storing in desiccated, sealed container. In context to the present study, the Autsralian Synchroton was used.

Studying the morphology of the dNPs formulated

In order to determine the potentiality of the sugar/polyols for dNPs,  sugar/polyol and CMC with varying w/w ratio were fabricated (Figure-2). Previous reports suggested that in case of only CMC, dNPs formed uniform structures (Figure-2a). In contrast to the given phenomena, addition of sucrose to the CMC resulted in the formation of brittle, clear, glassy material dNPs (Figure-2b). It has also been observed that an increase in the concentration of the sucrose resulted in the formation of more brittle dNPs. A similar trend was also observed in case of trehalose, which also resulted in formation of brittle dNPs (Figure-2c). However keeping the w/w ratio of trehalose: CMC as low as 2.5:1, resulted in formation of large opaque crystal dNPs (Figure 2c inset). Thus, addition of trehalose resulted in changing the normal morphology of the dNPs.


On the other hand, the addition of sorbitol to the CMC formulation resulted in the formation of clear and highly malleable dNPs with varying macroscopic properties (Figure 2d). However with the increase in the concentration of sorbitol, the malleability of the dNPs produced decreased though the dNPs did not become completely rigid and the same morphological projection was maintained at all w/w ratio of sorbitol: CMC.

In case of mannitol, the addition of the sugar to the CMC resulted in formation of large crystals at all w/w ratios (even at 1:1 ratio), thus no suitable dNP projections were observed, and hence mannitol was excluded from the given experiment.

Figure-2.Images showing the morphological features of the dNPs: a) Uniform solid dNPs was   produced in case only CMC b) Brittle dNPs produced in case of sucrose/CMC c) Very brittle dNPs produced in case of trehalose/CMC d) Ductile and highly malleable dNPs formed in case of sorbitol/ CMC

In order to verify that CMC sugar/polyp formulation was not displaced, 3000 MW fixable rhodamine-dextran was added to the respective sugar/polyp formulation. The morphology of the resultant dNPs was then observed by analyzing under a fluorescence confocal microscope, which helped in verifying the rhodamine-dextran formulation. It was observed that the rhodamine-dextran mixture remained within the projection for each formulation case (Figure 3 a-d). The dNPs thus produced were applied to the skin and arrows indicated the projections (Varughese et al. 2013).

Thus, it can be clearly stated that the addition of the sugar/polyols into the CMC lead to the formation of dNPs with significant morphological and physical changes in comparison to the dNPs produced in case of pure CMC (Davey, Schroeder and ter Horst, 2013).

Figure-3.Fluroscence confocal images of dNPs fabricated with rhodamine dextran and different CMC/sugar formulations a) Only CMC b) 10:1 w/w ratio of sucrose to CMC c) 10:1 w/w ratio of trehalose to CMC d) 30:1 w/w ratio of sorbitol to CMC

The present analysis helped in determining the crystalline nature of the dNP formulations (Winick and Doniach, 2012). Crystalline structures resulted in formation of defined peaks. The pure CMC samples did not produce any suitable peak and hence are considered amorphous (figure 4a). However, crystalline peaks were observed when sorbitol was added to formulation (figure 4b) but at a specific ratio of 3.3:1, no such crystalline peaks were detected. This indicated that decrease in the concentration of sorbitol resulted in overlapping of the peaks (Liu et al. 2015). In case of trehalose two distinct peaks were observed (figure 4c) while in case of sucrose no such peaks were observed at any w/w ratio (figure 4d)

Figure 4.FT-FIR analysis of the CMC’s. a) CMC (no peaks) b) Sorbitol and CMC c) Trehalose and CMC

d) Sucrose and CMC

Three distinct failures of projection were observed which were analyzed using SEM (figure 6) (Sohda et al. 2014). Failure in bending of the tip was observed due to initial bending of the tip (figure 6 b, c, and f). Failure due to buckling was observed in cases where projections show that the material has bent (figure 6a). Finally bending of the projection over one another resulted in failure due tp brittleness (figure 6 d, g and h).

Formulation of dissolving Nanopatches (dNPs)

Figure 6.SEM images showing of the projections.  a)Only CMC b) 1:1 sucrose-CMC c) 10:1 sucrose-CMC d) 1:1 trehalose-CMC e) 10:1 trehalose-CMC f)1:1 sorbitol: CMC g) 5:1 sorbitol: CMC h) 30:1 sorbitol: CMC [all in w/w ratio]

Application of Nanopatch technology provides a significant benefit over the old conventional methods of vaccination. The Nanopatch technology is associated with the introduction of first efficient and specific needle-free administration of the vaccine to specific cells, which evokes a rapid immunological response in response to a particular disease. Discussion on the present topic includes an understanding of the designing framework of the nanopatches. The Nanopatches are designed in such a way that it resembles a small chip primarily made up of gold-silicon wafer, which is lined with numerous numbers of small needles over which the vaccines are coated. Thus, nanopatches help in delivering an effective volume of the required vaccine to the respective target cells.

To understand the designing framework of the respective nanopatches in association with the medical world, the formulation process associated with the manufacture of the dNPs needs to be carefully studied. The technological aspect of the nanopatches plays a vital role in developing insight knowledge regarding the associated methods and biophysical techniques involved in the formulation process. Discussion on the given topic primarily emphasizes on the interpretation of the results observed by the given article. The results obtained helped in analyzing the change in the morphology of the respective dNPs produced in association with the sugar/polyol CMC with supporting data from the respective FT-FIR data and SEM images.

Formation of the dNPs from only CMC do not lead to the formation of uniform reliable structure, but contrastingly addition of various sugar/polyol to the CMC produced a marked change in the structural orientation of the respective dNPs thus produced. The addition of different sugar/polyol resulted in the formation of different kinds of dNPs. This suggests that addition of different sugar molecules produces a marked effect on the resultant dNPs formed. The concentration of the sugar/polyp also plays a vital role in producing a change in the structure of the resultant dNPs. While the addition of sucrose at any given w/w ratio resulted in the formation of brittle dNPs, decreasing the trehalose: CMC ratio resulted in the formation of large opaque crystalline structure. Hence, it can be stated that trehalose at low concentration is able to crystallize with a large number of nucleation event, which results in the growth of crystals and thereby contributes to the change in the morphology of the dNPs. On the other hand, sorbitol addition to the CMC resulted in the formation of completely different type’s dNPs, which were highly malleable. Significantly, it was observed that with the decrease in the concentration of sorbitol, the malleability increased and, therefore, it can be inferred that addition of sorbitol to the CMC resulted in adding a new dimension to the formed dNPs.

The physical and morphological changes thus produced in the resultant dNPs was observed by using a fluorescence dye rhodamine-dextran. Rhodamine-dextran being fluorescent in nature helps in determining the amount of sugar/polyp being incorporated on the surface of each dissolving dNPs. Rhodamine-dextran binds to the sugar/polyol molecule and thus helps in determining the particular sugar/polyol that has been incorporated in the given formulation.


The qualitative nature of the resulting dNP formulation is determined with the help of FT-FIR and thus helps in understanding about nature (amorphous or crystalline) of the resulting dNP formulations. A presence of defined peaks contributed to the crystalline nature of the formulation while the absence of such peaks suggests that the formulations are amorphous in nature. Thus after interpretation of the results, it can be discussed that the pure sample of CMC never formed defined peaks and hence are considered amorphous. While the addition of the corresponding sugar/polyp in to the CMC resulted in the formation of dNPs that produced defined peaks. Hence, the addition of the different sugar/polyol residues contributes to the crystalline nature of the resulting dNP formulation and depending upon the w/w ratio the nature of the dNP formulation changes accordingly. Although the addition of various sugar/polyol residues contributed to the crystalline nature of the resulting dNP, it is also considered that with the change in the w/w ratio the nature of the formulation also changed. For example, the addition of sorbitol resulted in the presence of defined peaks while at a ratio below 3.3:1 no real peaks were observed. Similar trends were also observed in the case of the other sugar residues, which resulted in the formation of a high peak at given ratio, and with the decrease in the ratio, no such peaks are observed.

Thus, the FT-FIR data analysis helped in validating the qualitative results. It can be also stated that those sugar molecules that are already crystalline in nature o not produce any peaks in the case when they were incorporated into the CMC and as a result the dNPs produce are amorphous in nature. Hence, the analysis provided accurate and reproducible information regarding the intermolecular interactions (amorphous and crystalline properties) of the complex heterogeneous (sugar-CMC) solid formulations.

The method of Nanoindentation helped in providing information regarding the bulk properties of the associated dNPs formulations. Single projections were measured by applying a particular force, which helped in studying regarding the surface property of the formulated dNPs. Finally, it can be stated that the various kinds of projections observed under SEM produced images that help in the understanding of the distinct failures, which resulted in causing a deflection. The deflection produced primarily emphasizes on the projection that arises due to failure in bending of the tip, failure due to buckling and failure due to brittleness.


Corrie, S., Depelsenaire, A. and Kendall, M., 2012. Introducing the nanopatch: a skin-based, needle-free vaccine delivery system. Australian Biochemist, 43(3), pp.17-20.

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