The authors have declared that no competing interests exist.
Planned and prepared the vacuum deposition experiments: ESZ VK RA. Performed optical: ESZ YKM RA. Measurements: AFM SEM. Planed and performed electrical characterizations: DG. Provided pharmaceuticals: HS FHF RS. Planed and performed Raman measurements and biological experiment: HS FHF RS. Selected and provided medical grade implant substrates and the medical application background: BGS. Wrote and discussed the article: RA YKM ESZ RS FHF HS BGS.
Nanostructuring of drug delivery systems offers many promising applications like precise control of dissolution and release kinetics, enhanced activities, flexibility in terms of surface coatings, integration into implants, designing the appropriate scaffolds or even integrating into microelectronic chips etc. for different desired applications. In general such kind of structuring is difficult due to unintentional mixing of chemical solvents used during drug formulations. We demonstrate here the successful solvent-free fabrication of micro-nanostructured pharmaceutical molecules by simple thermal evaporation (TE). The evaporation of drug molecules and their emission to a specific surface under vacuum led to controlled assembling of the molecules from vapour phase to solid phase. The most important aspects of thermal evaporation technique are: solvent-free, precise control of size, possibility of fabricating multilayer/hybrid, and free choice of substrates. This could be shown for twenty eight pharmaceutical substances of different chemical structures which were evaporated on surfaces of titanium and glass discs. Structural investigations of different TE fabricated drugs were performed by atomic force microscopy, scanning electron microscopy and Raman spectroscopy which revealed that these drug substances preserve their structurality after evaporation. Titanium discs coated with antimicrobial substances by thermal evaporation were subjected to tests for antibacterial or antifungal activities, respectively. A significant increase in their antimicrobial activity was observed in zones of inhibition tests compared to controls of the diluted substances on the discs made of paper for filtration. With thermal evaporation, we have successfully synthesized solvent-free nanostructured drug delivery systems in form of multilayer structures and in hybrid drug complexes respectively. Analyses of these substances consolidated that thermal evaporation opens up the possibility to convert dissoluble drug substances into the active forms by their transfer onto a specific surface without the need of their prior dissolution.
Important tasks for pharmaceutical engineering are the molecular scale mixing and structuring of pharmaceutical drugs for increased solubility. This includes coating of implants for multifunctional applications, or creation of hybrids/multilayers from different drugs and carriers for designing the controlled and sequential release. However, due to the nature of fluids this is difficult to achieve with conventional solvent based methods. Furthermore, the vast majority of newly discovered drugs result in molecules of poor solubility and currently only less than 10% of new drug substances have both high solubility and permeability
From an engineering point of view the control over the drug particle size is very essential, as a smaller particle size (particularly in nanodimensions) leads to higher rates of dissolution
In this work, we introduce a versatile approach for formulating micro-nanostructured solvent free drug delivery systems in various forms by using conventional physical vapour deposition technique. Thermal evaporation (TE) processes
In order to evaluate the suitability of thermal evaporation for pharmaceuticals, we tried the approach on several (>30) pharmaceutical substances (
a) Raman spectra of the deposited and reference acetyl salicylic acid (ASS), showing clear Raman peaks in the TE deposited ASS. 3D AFM image in the figure a, from the TE ASS sample, shows the micro-crystallites of ASS. b–e: The antimicrobial activity of Erythromycin (b: control, c: TE deposited) against Staphylococcus aureus and of Itraconazole (d: control, e: TE deposited) against Candida albicans demonstrated in agar diffusion tests. The activity is significantly increased in the case of specimen deposited by TE. f) Tuneable crystallinity: 3D Atomic force microscope image of Nipasol grown on Ti substrate at room temperature resulting in a much larger crystal size as compared to that deposited at −100°C as shown in g) which is nano-crystalline. h) & j) SEM images of nanostructured Cholesterol h) and Tetracaine-HCl j) drugs respectively fabricated on different substrates by thermal evaporation showing nanoscale spikes, see insets. The nanospike or platelet like geometries result from the growth kinetics.
Test Material; |
Melting point at atmosphere (typical evaporation temperature range) [°C]} | Deposition remarks | Maximum extent of inhibition area (cm2) [TE deposited on titan plates] | Maximum extent of inhibition area (cm2)[Control (paper, 30 µg/disc)] |
150 (∼90–118) | Crystalline deposits | 2.55 (S.* aureus); Amount<30 µg | 1.36 | |
|
190–193 (∼100–137) | Amorphous and crystalline deposits; Raman spectra | 3.11 (S. aureus); Amount<30 µg | 1.38 |
159–163 (∼100–194) | Crystalline deposits; Raman spectra | 2.06 (S. aureus); Prove of prin-ciple, amount not further quantified | 1.67 | |
95–98 (∼85–165) | Crystalline deposits | 1.16 (S. aureus); Prove of prin-ciple, amount not further quantified | Under progress | |
|
250–260 (∼160–300) | Amorphous and crystalline deposits | 0.57 (S. aureus) Amount<30 µg | 0.68 |
215–220 (∼150–250) | Amorphous and crystalline deposits | 3.95 (S. aureus) Amount<30 µg | 1.64 | |
170–175 (∼100–223) | Crystalline deposits | 1.44 (S. aureus) Amount∼3.25 µg | 1.82 | |
220–223 (∼119–255) | Crystalline deposits | 1.65 (S. aureus) Amount<30 µg | Under progress | |
185 (∼100–197) | Amorphous and crystalline deposits | 0.67 (S. aureus) Amount<30 µg | 0.80 | |
147–149 (∼89–164) | Crystalline deposits; Raman spectra | 1.76 (Candida albicans); Proveof principle, amount notfurther quantified | 1.72 | |
166,2 (∼100–232) | Amorphous and crystalline deposits | 1.82 (Candida albicans) Proveof principle, amount notfurther quantified | 0.97 | |
146 (∼88–226) | Amorphous and crystalline deposits | 2.83 (Candida albicans)Amount <30 µg | 1.07 | |
167–171 (∼110–215) | Crystalline deposits/Raman Spectra | |||
200–203 (∼117–250) | Crystalline deposits/Raman Spectra | |||
148–150 (∼90–165) | Crystalline deposits | |||
224–226 (∼ 150–300) | Amorphous deposits | |||
|
283 (∼115–250) | Crystalline deposits | ||
200–203 (∼110–250) | Crystalline deposits | |||
135 (∼90–140) | Crystalline deposits | |||
168 (∼97–130) | Crystalline deposits | |||
284 (∼135–270) | Crystalline deposits/Raman spectra | |||
190–192 (∼123–200) | Crystalline deposits | |||
149 (∼114–220) | Crystalline deposits | |||
199–203 (∼100–170) | Amorphous and crystalline deposits | |||
155 (∼150–190) | Amorphous and crystalline deposits | |||
225–230 (∼160–200) | Amorphous and crystalline deposits | |||
152.1(∼119) | Crystalline deposits | |||
227–228 (∼120–250) | Crystalline deposits | |||
|
223 | Deposition not possible | ||
49–53 | Deposition not possible (decomposed to gas) | |||
134–136 | Decomposes, droplets on the surface of substrate | |||
56–58 | Deposition not possible (Degrades to dioxin) |
First part (1–12) lists the materials which has been successfully nanostructured by thermal evaporation and have been tested with disk diffusion method. The second part (13–27) lists the successfully deposited materials however the biological tests are under progress. The last part (28–31) lists the pharmaceutical substances which are not suitable for thermal evaporation as they are decomposed during deposition. (*Staphylococcus).
In view of the variety of molecules being capable of thermally evaporated found here, there seems to be no regularity which of the complex molecules stay intact and which decompose. A general precondition for TE process is that the thermal energy (here >300°C meaning ∼0.05 eV) injected into the molecules, stays far away from the range of the intermolecular binding energies of typically 2–7 eV, which seems to be fulfilled. For a better comparison of the process, it should be understood in which energy steps a single molecule can leave the different bonding force fields in its surrounding liquid and enter the vapor phase, or otherwise which energy barriers have to be taken in sequence to decompose a molecule. Such sophisticated calculations are difficult to carry out and not yet known, the large variety of possibilities for the molecules to wiggle, swing or rotate in its liquid state complicates it to foresee which of the molecules find their way into the gas phase and which decompose. Even if the decomposition occurs in some fragments a recomposition on the substrate could occur. Our arbitrary choice of molecules seems to confirm this, but an obvious prediction of the suitability for TE needs significant efforts to be done in future. It must be noted that relatively large molecules like Pindolol, Erythromycin or Tetracaine-HCl also remain intact as confirmed by Raman investigations. In principle, the evaporation of the molecules may have a positive side effect by purifying the evaporated substance, as the contaminants with a lower melting point are evaporated early when the substrate is still covered by a shutter of the evaporation oven. Other way round those contaminants higher melting point will not evaporate at all.
With the molecules being suitable for evaporation, the full versatility of thermal evaporation can be utilized. This means that by changing the deposition rate, the crystallinity of the deposited film can be controlled in a wide range from amorphous to almost single crystalline.
The inset images inside
A lateral structuring i. e. the positioning of thermal evaporated thin drug films, is relatively straight forward by utilizing lithography techniques or shadow masks. Possible applications for laterally structured systems are smart or multifunctional pharmaceutical drug coatings for implants or contact lenses. Specific examples are contact lenses for drug delivery
a) Representative SEM images of microstructured ASS morphology by deposition through a microscopic shadow mask. b) magnified SEM image of the square shaped of the deposited drug. c): current voltage response of a pharmaceutical field effect transistor (PFET) of Pilocarpine-HCl, cross section scheme (upper left) and part of the waver (lower right) used for the lateral structuring of the Pilocarpine-HCl PFET fabrication are shown as insets.
Additionally, the appropriate fabrication of multilayers of pharmaceuticals controls the release kinetics of the drug which can find potential applications in drug deliveries etc. A fast dissolving drug can be protected by a second layer that needs to be dissolved first. Just for demonstration 2-layered structure using Metronidazole and PLGA on titanium substrate was fabricated by thermal evaporation technique and its release kinetics was studied with respect to incubation time which is shown in
a) Comparative dissolution profile of Metronidazole alone and Metronidazole covered with the PLGA thin layer. The insets (i) and (ii) in the fig. 4a show the schematic of the multilayer coating and SEM image of the TE deposited Metronidazole drug respectively. b) 3D SEM micrograph an a cross section cut in the structure of a double layer showing top layer of Tetracaine and second layer of Metronidazole deposited on Ti substrate, c) SEM Image of the nanocomposite of Tetracaine-HCl with Ag on the Si substrate fabricated by co-deposition from two sources simultaneously sources and d) SEM image of Tetracaine-HCl without silver for comparison.
Another very promising route of a new drug design by thermal evaporation is the co-deposition of two different drugs from two separate evaporators simultaneously in a vacuum chamber. This allows the formation of a molecular scale mixing of pharmaceutical active substances or excipients to form nanostructured hybrid drugs which can exhibit synergetic responses at the same time. With conventional solution based processing, such a mixing requires very special preconditions, e.g., the possibility for co-crystallisation. Evaporation of molecules from two different sources typically gives no time for a demixing process on the substrate, as molecules directly loose most of their energy when they hit the substrate surface. Here, even amorphous mixtures can be formed with structurally incompatible drugs, leading to synergistic effects. The approach is very beneficial for controlling the dissolution of drugs and furthermore the solubility can also be significantly increased. If a molecule with poor ability to dissolve is only surrounded by molecules of good solubility, a total increase in dissolution can be effected. This is very beneficial in increasing the solubility of poorly soluble drugs by deposition of a low soluble molecules in parallel with a highly soluble one and with this forming a solid solution. The co-deposition approach also allows the creation of a molecular mixture in terms of a solid solution from nearly every thermally evaporated drug (overviewed
A completely new way of pharmaceutical engineering is the combination of inorganic materials with pharmaceutical active substances. Such combinations could be potentially used as smart implant coatings that form nanoporous structures with medication filled pores in the inorganic material. In terms of the oral implant example given above, these molecules may be embedded in nanoscale sponge like ceramic structures by co-evaporation to have a potential depot function of antibacterial factors in order to reduce the risk for periimplantitis. An example for such co-evaporation is shown by SEM measurement in
In summary, thermal evaporation of pharmaceutical molecules can be utilized as a completely solvent free technique where drugs as powders or crystalline materials are directly formulated into desired nano or micro structures and shapes on preferred substrates. The performed deposition experiments and their structural analyses suggest that by thermal evaporation technique most of pharmaceutical substances (>80%) can be micro-nanostructured in different desired complex forms. The observed features of enhanced solubility and activity, controlled release kinetics of synthesized drugs seem to be a very powerful alternative for the traditional physical structuring of pharmaceutical drug molecules. The variety of new possibilities for pharmaceutical engineering utilizing thermal evaporation is expected to be very wide and will be very beneficial for many branches of medicine, pharmacy and engineering. Since a large number of drugs are accessible to the approach, thermal evaporation tests could result in reduction of dose and side effects for many approved drugs. The possibility of smart designing like hybrid, multilayer and/or implant coatings or solid solutions could lead to a re-assessment of insoluble molecules and thus to could be very promising for many new therapies. Here we have demonstrated that thermal evaporation technique can be versatily employed for micro-nanostructuring of different pharmaceutical drugs in various desired forms with increased responses. As an outlook, we would like to emphasize that thermal evaporation is not only limited to the drugs listed here but also can be employed for restructuring of unrestricted old pharmaceutical into new forms of medications with improved responses.
Active pharmaceutical ingredients and excipients selected for this study were Nipasol, Tetracycline-HCl, Tetracycline hydrate, Chloramphenicol, Pilocarpine-HCl, Pindolol, Clotrimazole, Diclofenac Sodium, Erythromycin (base), Metronidazole, Itraconazole, Neomycin sulphate, Novobiocin Sodium, Vancomycin-HCl, Sulfathiazole, 5-Fluorouracil, Acetyl salicylic acid (ASS), Paracetamol, Trimethoprim, Indometacin, Tetracaine-HCl, Caffeine, Ascorbic acid and excipient materials and biodegradable polymers like Cholesterol, Adepic acid and Poly-lactic-co-glycolic acid (PLGA) and Polyglycolic acid (PGA) which are all commercially available and were purchased by common suppliers (Merck, Sigma Aldrich).
Thin films of active pharmaceutical materials were deposited by thermal evaporation in a vacuum chamber evacuated by a turbo molecular pump to 10−5 to 10−7 mbar on various substrates kept at room temperature and −100°C. The thermal evaporators are of Knudsen type equipped with metal or graphite crucibles. Deposition temperatures vary for different drugs and hence several drugs are deposited at several temperatures (see
Bright field optical micrograph images of deposited drugs on different substrates were taken by Zeiss optical microscope equipped with a Leica DFC 280 digital camera system at different magnifications. Atomic force microscopy (AFM) images were recorded with Thermo Microscope-Veeco autoprobe AFM equipped with an optical unit and electronic module in contact and non contact mode. Standard silicon nitride tips mounted on cantilevers were used. Philips-FEI XL30 SEM equipped with an EDAX EDX detector was used to analyse the structural geometry of nano- or micro- scale deposited drugs. In order to avoid the structural damage in the structures and the charging, acceleration voltages were kept as small as possible down to 2 kV and reduced currents were applied. In order to get the actual morphologies of thermal evaporated pharmaceuticals, no further coating (of carbon or gold thin film) was performed on deposited drugs. This was the reason that the SEM images with higher magnification appear less focused. Raman spectra of raw drugs as well as thermally evaporated layers on different substrates were recorded to ensure the identity of substance using a Senterra Raman Microscope (Bruker Optik GmbH, Bremen, Germany) with OPUS software (Bruker). For the raw material, the sample was prepared on a sample holder or on a mirror plate to obtain a flat sample surface for measurement. The sample was mounted under the objective and the sample surface was brought in focus using the light microscopic image. For all measurements, a 20x magnification is identified as suitable and in combination with a slit aperture of 50×1000 µm ensures collection of the highest possible Raman intensity. Collection of the Raman spectra has been performed in the dark field with 785 nm laser excitation (50 mW) at a resolution of 3–5 cm−1 resulting in a full spectrum from 80–3500 cm−1 with 10 seconds integration time and 2 co-additions. The Raman shift was calibrated automatically using the SURECAL option of the instrument. For analysis, the Raman band positions in the fingerprint region of the vacuum deposited drugs were compared with respect to the Raman spectrum of the raw material.
Current-voltage (I-V) field effect transistor (FET) response of a nanostructured pharmaceutical drug fabricated directly on a microchip with gold contacts was measured by FET using a Keithley 6485 pico-amperemeter (optimised for measuring small signals) at different gate voltages. The source and drain contacts were fabricated by deposition through a shadow mask, the gate contact was realised in the substrate consisting of a p++ doped silicon wafer covered with a 100 nm thick gate oxide.
Preservatives, antibiotics and antimycotics deposited on round titan plates (Ø 1.0 cm, thickness 0.04 cm) were examined on their antimicrobial efficacy by means of the agar diffusion test. The test organisms, Staphylococcus aureus ATCC 6538 and Candida albicans ATCC 10231, were purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Muller Hinton Agar Medium, Sodium Chloride Peptone Broth, buffered (SCPB), Tryptic Soy Broth and Tryptic Soy Agar Medium were purchased as dehydrated powders from Merck (Darmstadt, Germany).
The test media were rehydrated with demineralised water and sterilised according to instructions of the supplier. For Candida albicans the Muller Hinton Agar was supplemented with 2% glucose monohydrate prior to sterilisation. After sterilisation and cooling down to approximately 50°C, the agar medium was poured into Petri dishes (Ø 9 cm) in aliquots of 10 ml. Into the coagulating medium titanium plates covered with vacuum deposited antimicrobial substances were placed on their sides in an upright position. Each titanium plate was deposited in the middle of the agar layer of a Petri dish.
Test suspensions of Staphylococcus aureus and of Candida albicans were prepared from overnight cultures at 34°C by floating the cultures using sterile sodium chloride peptone buffer. The cell concentrations in the resulting suspensions were assessed by means of a spectrophotometer and the suspensions were diluted in order to adjust microbial numbers of 5×105 cfu/ml. 0.1 ml-aliquots of the suspensions were used to inoculate 5 ml portions of sterilized and cooled agar medium to achieve test concentrations of about 104 colony forming units per ml. Immediately after inocculation and mixing, these portions of the fluid agar medium were poured into the test dishes in order to get homogenous layers around the titan plates. Then the agar was allowed to gelate.
Control plates were prepared using paper discs (Ø 0.6 cm) which were impregnated with 20 µl of a diluent containing the required test substance. In the case of poor water solubility dimethylsulfoxide was used, hydrophilic substances were dissolved in sterile demineralised water. As final concentration per paper disc, 30 µg of the respective substance were chosen. The discs were placed on the surfaces of inoculated agar medium plates after their gelation. The test Petri dishes were incubated at 35°C for 20 hours and then the extents of the inhibition areas were measured.
The authors gratefully acknowledge several grants: E. S. Z. a Ph. D. grant from Egyptian government, Y.K.M. a postdoctoral grant from Alexander von Humboldt foundation and R. A. a Heisenberg Professorship by the Deutsche Forschungsgemeinschaft (DFG). Furthermore, R. A. wants to thank the DFG founded Inflammation at Interfaces cluster for its support.