Patent Application
20230130513
The present invention relates to the fields of dentistry, oral diseases, and drug delivery systems. More particularly, the present invention relates to microemulsions as magnetic responsive nanoplatforms for the delivery of formulations to pathogenic microbes in the oral environment during photodynamic therapy.
Biofilm control within the complexity of the intraoral environment is a challenging task (1). The continuing rise in antibiotic resistance, the ongoing problem with patient compliance, and the difficulty in eradicating biofilms demand the use of combinatorial strategies (2). Biofilm formation is an important virulence mechanism of oral pathogens (3). Oral dysbiotic biofilms trigger major oral diseases, such as dental caries, periodontitis, and endodontic infections (4). Caries-related pathogens can secrete a mixture of polysaccharides to construct and maintain a structured multicellular bacterial community into biofilm to survive and grow (5). The extracellular matrix resists antimicrobial agents from reaching targeted microbial cells by diffusion limitation. This barrier for tolerance against antimicrobial agents has been recognized (6,7).
Antimicrobial photodynamic therapy (aPDT) is a promising adjunctive method for modulating pathogenic oral biofilms that is well supported by a plethora of literature (7-10). Antimicrobial photodynamic therapy uses non-toxic dyes called photosensitizers (PS) that can be excited by harmless visible light to produce cytotoxic reactive oxygen species (ROS) (5). Antimicrobial photodynamic therapy involves a multi-stage process, including topical photosensitizer administration, light irradiation, and interaction of the excited state with the surrounding oxygen (11). For oral biofilm-associated diseases, especially dental caries, antimicrobial photosensitizers based on cationic phenothiazine dye, toluidine blue O (TBO), have been well studied and mediated expressive photodynamic results (11,12). However, some difficulties still need to be improved in antimicrobial photodynamic therapy, such as specific targeting and biological compatibility.
Clinically, antimicrobial photodynamic therapy performance is confined to subsidiary outcomes without clinically relevant reduction greater than 3-log (>99.9%) (11,13,14). The conceivable explanations for the resistance of biofilms are limited diffusion and interaction of antimicrobial agents through the biofilm, altered levels of metabolic activity within the biofilm, and genetic adaptation (5). In addition, the transport and infiltration of the photosensitizer into the biofilm core structure is an essential step in optimizing the antimicrobial photodynamic therapy performance (15).
Several antibiofilm strategies have been explored to modulate biofilm formation and development (16). In addition, targeting strategies to break the biofilm barrier or enhance the infiltration of antibacterial agents are being increasingly explored. Therefore, the rational design of photosensitizer-based nanoplatforms to overcome the above key obstacles for achieving potent aPDT is of great significance for managing oral infections.
The prior art is deficient in antimicrobial photodynamic therapies utilizing a magnetic-responsive photosensitizer-based nanoplatform. The present invention fulfills this long-standing need and desire in the art.
The present invention is directed to an antimicrobial microemulsion. The microemulsion comprises a plurality of superparamagnetic iron oxide nanoparticles (SPIONs) and a photosensitizer in a functional association with the plurality of SPIONs.
The present invention is further directed to a method for treating an oral disease in a subject in need thereof. In this method, the antimicrobial microemulsion described herein is applied to an oral area of interest in the subject. The plurality of superparamagnetic iron(II) oxide nanoparticles and the photosensitizer in the functional association therewith are irradiated during an application of a magnetic field to the superparamagnetic iron(II) oxide nanoparticles. The plurality of superparamagnetic iron(II) oxide nanoparticles and the photosensitizer are targeted to the oral area of interest via the magnetic field, thereby treating the oral disease in the subject.
The present invention is directed further to a magnetic-responsive photodynamic nanoplatform. The magnetic-responsive photodynamic nanoplatform comprises, in a microemulsion, a plurality of superparamagnetic iron(II) oxide nanoparticles and an organic photosensitizer functionally associated with the plurality of superparamagnetic iron(II) oxide nanoparticles.
The present invention is directed further still to a process for decreasing a microbial population in a pathogenic oral biofilm. In this method, the oral biofilm is contacted with the microemulsion comprising the magnetic-responsive photodynamic nanoplatform described herein. A photodynamic therapy is applied to the plurality of superparamagnetic iron(II) oxide nanoparticles and the organic photosensitizer functionally associated therewith in the microemulsion. A magnetic field is applied to the superparamagnetic iron(II) oxide nanoparticles during the photodynamic therapy to target the plurality of superparamagnetic iron(II) oxide nanoparticles and the organic photosensitizer to the microbial population of the pathogenic oral biofilm, where the plurality of superparamagnetic iron(II) oxide nanoparticles and the organic photosensitizer are pharmacologically effective to decrease the microbial population therein.
The present invention is directed further still to a method for improving the efficacy of a photosensitizer during an antimicrobial photodynamic therapy (aPDT) treatment of oral diseases. In this method, a photosensitizer and a plurality of superparamagnetic iron(II) oxide nanoparticles are encapsulated in a microemulsion whereby the photosensitizer is functionally associated with the plurality of superparamagnetic iron(II) oxide nanoparticles. The photosensitizer and the superparamagnetic iron(II) oxide nanoparticles are irradiated to activate the same during an application of a magnetic field thereto, whereby the magnetic field directs the superparamagnetic iron(II) oxide nanoparticles with the functionally associated photosensitizer to a pathogenic microbe causing the oral disease, thereby improving the efficacy of the photosensitizer during the aPDT treatment of the oral disease.
Other and further aspects, features, benefits, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
The appended drawings have been included herein so that the above-recited features, advantages, and objects of the invention will become clear and can be understood in detail. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.
As used herein, the term “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method described herein can be implemented with respect to any other method described herein.
As used herein, the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used herein, “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps unless the context requires otherwise. Similarly, “another” or “other” may mean at least a second or more of the same or different claim element or components thereof.
As used herein, “photosensitizer-based nanoplatform”, “magnetic-responsive photodynamic nanoplatform”, “nanoplatform”, “antimicrobial microemulsion”, and “MagTBO nanoplatform” are used interchangeably.
As used herein, “contacting” refers to any suitable method of bringing the superparamagnetic iron oxide nanoparticles and the photosensitizer comprising the nanoplatforms or antimicrobial microemulsions described herein into contact with a pathogenic oral biofilm or pathogenic microbe associated with an oral disease. For in vivo applications, any known method of administration within the oral cavity is suitable. In vitro, this is achieved by exposing the pathogenic oral biofilm or pathogenic microbe to the superparamagnetic iron oxide nanoparticles and the photosensitizer in a suitable medium.
As used herein, “pharmacologically effective” refers to an amount or concentration of the superparamagnetic iron oxide nanoparticles and the photosensitizer in the microemulsion that results in an improvement or remediation in the oral disease or in a decrease in or a reduction up to elimination of the microbial population of a pathogenic oral biofilm. A person having ordinary skill in this art would understand that the pharmacologically effective amount may improve the patient's or subject's condition, but may not be a complete cure of the oral disease or effect a complete elimination of the microbial population in the oral biofilm.
As used herein, the term “subject” refers to any target or recipient of the treatments utilizing the magnetic-responsive photodynamic nanoplatforms or antimicrobial microemulsions provided herein.
In one embodiment of the present invention there is provided an antimicrobial microemulsion, comprising a plurality of superparamagnetic iron oxide nanoparticles; and a photosensitizer in a functional association with the plurality of superparamagnetic iron(II) oxide nanoparticles.
In this embodiment, the superparamagnetic iron oxide nanoparticles may be superparamagnetic iron(II) oxide nanoparticles. Also in this embodiment a representative photosensitizer is toluidine blue O (TBO). The antimicrobial microemulsion may comprise a magnetic-responsive photodynamic nanoplatform configured for targeted delivery of the plurality of superparamagnetic iron(II) oxide nanoparticles and the photosensitizer.
In a related embodiment, the present invention provides a pharmaceutical composition comprising the antimicrobial microemulsion, as described supra.
In another embodiment of the present invention, there is provided a method for treating an oral disease in a subject in need thereof, comprising applying the antimicrobial microemulsion, as described supra, to an oral area of interest in the subject; irradiating the plurality of superparamagnetic iron(II) oxide nanoparticles and the photosensitizer in the functional association therewith during an application of a magnetic field to the superparamagnetic iron(II) oxide nanoparticles; and targeting, via the magnetic field, the plurality of superparamagnetic iron(II) oxide nanoparticles and the photosensitizer to the oral area of interest, thereby treating the oral disease in the subject.
In an aspect of this embodiment, the irradiating step may comprise applying an antimicrobial photodynamic therapy. In this embodiment and aspect, thereof the oral disease may be selected from the group consisting of at least one of caries, periodontitis, peri-implantitis, and an endodontic infection.
In yet another embodiment of the present invention, there is provided a magnetic-responsive photodynamic nanoplatform, comprising, in a microemulsion, a plurality of superparamagnetic iron(II) oxide nanoparticles; and an organic photosensitizer functionally associated with the plurality of superparamagnetic iron(II) oxide nanoparticles. In this embodiment, the organic photosensitizer may be toluidine blue O (TBO).
In yet another embodiment of the present invention, there is provided a process for decreasing a microbial population in a pathogenic oral biofilm, comprising contacting the oral biofilm with the microemulsion comprising the magnetic-responsive photodynamic nanoplatform, as described supra; applying a photodynamic therapy to the plurality of superparamagnetic iron(II) oxide nanoparticles and the organic photosensitizer functionally associated therewith in the microemulsion; and applying a magnetic field to the superparamagnetic iron(II) oxide nanoparticles during the photodynamic therapy to target the plurality of superparamagnetic iron(II) oxide nanoparticles and the organic photosensitizer to the microbial population of the pathogenic oral biofilm, where the plurality of superparamagnetic iron(II) oxide nanoparticles and the organic photosensitizer are pharmacologically effective to decrease the microbial population therein.
In an aspect of this embodiment, the step of applying the photodynamic therapy may comprise irradiating the plurality of superparamagnetic iron(II) oxide nanoparticles and the organic photosensitizer functionally associated therewith. In this embodiment and aspect thereof the photodynamic therapy is an antimicrobial photodynamic therapy. In this embodiment and aspect thereof, the microbial population in the pathogenic oral biofilm may be a single pathogenic species biofilm or a multispecies pathogenic biofilm. In addition the microbial population may comprise at least one oral pathogenic microbe selected from the group consisting of Streptococcus mutans, Enterococcus faecalis, and Porphyromonas gingivalis.
In yet another embodiment of the present invention, there is provided a method for method for improving the efficacy of a photosensitizer during an antimicrobial photodynamic therapy treatment of an oral disease, comprising encapsulating a photosensitizer and a plurality of superparamagnetic iron(II) oxide nanoparticles in a microemulsion whereby the photosensitizer is functionally associated with the plurality of superparamagnetic iron(II) oxide nanoparticles; and irradiating the photosensitizer and the superparamagnetic iron(II) oxide nanoparticles to activate the same during an application of a magnetic field thereto, whereby the magnetic field directs the superparamagnetic iron(II) oxide nanoparticles with the functionally associated photosensitizer to a pathogenic microbe causing the oral disease, thereby improving the efficacy of the photosensitizer during the aPDT treatment of the oral disease.
In an aspect of this embodiment, the microemulsion encapsulating the superparamagnetic iron(II) oxide nanoparticles and the photosensitizer functionally associated therewith may comprise a photodynamic nanoplatform. In this embodiment and aspect thereof the photosensitizer may be toluidine blue O (TBO). In this embodiment and aspect thereof, the oral disease may be selected from the group consisting of at least one of caries, periodontitis, peri-implantitis, and an endodontic infection. In addition, the pathogenic microbe may be selected from the group consisting of at least one of Streptococcus mutans, Enterococcus faecalis, and Porphyromonas gingivalis.
Provided herein are magnetic-responsive, photosensitizer-based nanoplatforms and antimicrobial microemulsions which enable an enhanced antimicrobial effect via association with iron(II) oxide or ferrous oxide nanoparticles (Fe2O3) and magnetic field navigation. The magnetic-responsive photodynamic nanoplatform of the present invention (MagTBO) was constructed by assembling TBO and superparamagnetic iron oxide nanoparticles using a continuous microemulsion (F1).
In the nanoplatform, the synthesized microemulsions improved the stability, dispersity, and biocompatibility of nanoparticles and enhanced the antimicrobial action of the TBO photosensitizer. Moreover, the exposure to external magnetic forces allows the motion of the therapeutic agents toward deep sites inside the biofilms, providing potential disruption of the self-produced extracellular polysaccharide biofilm matrix and biofilm reduction. The present invention demonstrates that antimicrobial photodynamic therapy via the MagTBO nanoplatform, guided by magnetic force, are pharmacologically effective to treat biofilms formed by a major pathogen associated with dental caries and other oral diseases.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
The TBO photosensitizer has an absorption peak between 594 and 632 nm, assessed via ultraviolet-visible optical absorption spectrometry (SpectraMax M5, Molecular Devices, Sunnyvale, Calif.). The TBO spectrum also was assessed using Fourier-transform infrared spectroscopy (FTIR) (Nicolet 6700, Thermo Fisher Scientific, Waltham, Mass.). A light-emitting diode (LED; photoactivated disinfection (PADLight-F3WW, Beijing, China) with a narrow spectrum emission ranging from 664 to 670 nm was used for the light activation. The diameter of the LED light tip was 6 mm. A power meter Lasermate (Coherent Inc., Santa Clara, Calif.) at a 2 mm distance from the LED light tip was used to measure the peak power. The irradiation of 5 min was associated with an energy density of 180 J/cm2, which was calculated using the following equation:
where power density is:
Superparamagnetic iron(II) oxide nanoparticles were synthesized via chemical co-precipitation as described by Sun et al. (17). Briefly, 10 mL of deionized water was used to dissolve 0.2 g of polyglucose-sorbitol-carboxymethyl-ether (PSC). Then, 15 mL of water containing 0.06 g of FeCl3 and 0.03 g of FeCl2 was added. The mixture was cooled to 5° C., and 1 g of 28% ammonium hydroxide was added and stirred for 2 min. Finally, the mixture was heated at 80° C. for 1 h and purified using a 100 kDa membrane with five cycles.
The size and distribution of superparamagnetic iron(II) oxide nanoparticles were examined by transmission electron microscopy (TEM, FEI Tecnai T20, Hillsboro, Oreg., USA) with the Software Imaging System CCD camera (Gatan UltraScan 1000, Milwaukee, Wis., USA). The images were captured using 80 kV and magnifications of 26,000× and 42,000×. The samples were examined in the microemulsion form. ImageJ software was applied to measure the size of the nanoparticles. The histogram illustrated the size distribution using Sigma Plot software (SYSTAT, Chicago, Ill., USA). A semi-analytical solution with 98% accuracy (Ortner2020SoftwareX, Furlani1994IEEE) was used to calculate the magnetic fields. The calculation used a neodymium magnet in a cylindric shape and a diameter of 10 mm, length of 30 mm, and T1 magnetic B-field strength. A Fe3O4 nanoparticle with an average diameter of 8.4 nm was chosen and treated as magnetic dipoles in the calculation. The magnetic dipole moment of the nanoparticle was derived from magnetization curves.
Human gingival fibroblasts (HGF, ScienCell, San Diego, Calif., USA) were cultured using fibroblast medium (FM) supplemented with 2% fetal bovine serum, 1% fibroblast growth supplement, 100 IU/mL penicillin, and 100 IU/mL streptomycin (18 76). When the viability of the cells was above 90%, the cells were seeded in the wells of a 96-well plate (5,000 cells per well) and incubated for 24 h. In the following day, the medium was removed, and different concentrations of superparamagnetic iron(II) oxide nanoparticles (0.25, 0.5, 1, 1.5, 2, 2.5, and 5 wt. %) nanoparticles with and without 100 μg/mL of TBO photosensitizer were dissolved in the fibroblast medium and incubated in contact with the cells for 24 h. The synthesized microemulsions containing the different concentrations of superparamagnetic iron(II) oxide nanoparticles (0.25, 0.5, 1, 1.5, 2, 2.5, and 5 wt. %) were also tested. All the microemulsions were diluted with the fibroblast medium at a 1:1 ratio. After 24 h of incubation at 37° C. with 5% CO2, the cells were washed with phosphate-buffered saline (PBS), and cell counting Kit-8 (CCK-8, Dojindo, Rockville, Md., USA) was used to evaluate the cell viability. The absorbance was read at 450 nm using a spectrophotometer (SpectraMax M5, Molecular Devices, Sunnyvale, Calif., USA). For the live/dead assay, the fibroblast cells were seeded in the wells of a 24-well plate (40,000 cells per well). The medium was changed daily to allow the viability to be above 90%. Microemulsions containing 5 and 2.5% were added to a fresh culture medium at the ratio of 1:1 and incubated with the cells for one day. Wells treated with TBO alone or with no treatment were used as controls. After one day of incubation, the old medium was removed, cells were washed with PBS, and LIVE/DEAD™ Cell Imaging Kit (Invitrogen, Frederick, Md., USA) was used to stain the cells. The cells were visualized using an inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, N.Y., USA).
Three microemulsions were selected for further investigations (1, 2.5, and 5 wt. %). Microemulsions were tested for thermodynamic stability via centrifugation stress at 5,000 rpm for 2 h at 4° C., and freeze-thaw stress consisting of three complete cycles. Each cycle consisted of placing the microemulsion for 24 h at −20° C. followed by another 24 h at 4° C. The microemulsions were observed for any physical changes of phase separation (19,20). For long-term stability, microemulsions were kept in the dark at room temperature and then examined at 1, 3, and 6 months for physical changes or phase separation (21,22). The pH of the synthesized microemulsions was tested using a digital pH meter (accuracy±5%; Accumet XL25, Thermo Fisher Scientific, Waltham, Mass., USA). Before the pH measurement, the pH meter was calibrated using commercial standard buffer solutions of pH 4, pH 7, and pH 10 at room temperature. For the absorbance evaluation, the upper third of the synthesized microemulsions was investigated over time using ultraviolet-visible optical absorption spectrometry (SpectraMax M5, Molecular Devices, Sunnyvale, Calif., USA). A large change in the absorbance value indicates that the microemulsion may suffer from unbalanced distribution of the nanoparticles or some physical changes. The density of each microemulsion was calculated following the equation:
Photodynamic Treatment Via MagTBO Against Streptococcus mutans Biofilms
S. mutans UA159 obtained from the American Type Culture Collection (ATCC, Manassas, Va., USA) were grown overnight using brain heart infusion (BHI) broth (Sigma-Aldrich, St. Louis, Mo., USA) at 37° C. and 5% CO2 incubator. The optical density of the S. mutans was modified to 1×108 colony-forming units (CFU)/mL (OD600=0.9) and diluted 1:20 with BHI broth supplemented with 2% of sucrose (12). 200 μL of the diluted overnight culture was placed inside the wells of a black 96-well plate for 24 h. The media was changed after 24 h and 48 h of biofilm growth was continued. The biofilm was washed with 0.9% saline and irradiated with 100 μg/mL of TBO and MagTBO microemulsions containing 1, 2.5, and 5% of SPIONs with and without magnetic field (neodymium magnet, 0.4 Tesla; pull force=601b5). The TBO or the microemulsions were left for 1 min and then irradiated for 5 minutes to deliver an energy density of 180 J/cm2 and light intensity of 180 mW. The magnetic field was applied at a 10 mm distance below the biofilm during the entire period of the incubation and irradiation times (
After each treatment, the biofilms were washed by placing 200 μL of 0.9% saline inside the well, and then, the 0.9% saline was gently aspirated. Next, another 200 μL of saline was placed inside the well, and the biofilm was gently scraped to remove the biofilm cells (23). Then, the biofilm cells were resuspended with the saline to perform serial dilutions (1:10, 1:100, 1:1000, 1:10 000, and 1:100 000) using BHI agar. The BHI agar plates were incubated for 48 h at 37° C. in a 5% CO2 incubator and then counted using a colony counter. Considering the dilution factor, the CFUs/mL were calculated and estimated to measure the number of colonies per milliliter. Two wells were randomly selected for Live/Dead staining of the biofilms to be washed with 0.9% saline and then stained with the BacLight live/dead kit (Molecular Probes, Eugene, Oreg.). A combination of 2.5 μM SYTO 9 and 2.5 μM propidium iodide at a ratio of 1:1 was created to treat the cells for 10 min and observed using an inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, N.Y.).
Scanning Electron Microscopy (SEM) Imaging of the S. mutans Biofilms
To investigate the effect of the MagTBO microemulsion against the biofilm thickness and morphology, the 2.5% MagTBO effect against a 48-h S. mutans biofilms was further analyzed using Scanning Electron Microscope. S. mutans biofilms were grown over borosilicate glass slabs (VWR, Radnor, Pa., USA) for 48 h. Then, the biofilms were washed with PBS and fixed using 3% formaldehyde. On the following day, the biofilms were dried using ethanol dilutions followed by 100% hexamethyldisilazane. The biofilms were then sputter-coated with platinum and capture using SEM (Quanta 200, FEI Company, Hillsboro, Oreg., USA).
Photodynamic Treatment with MagTBO Against Saliva-Derived Biofilms
Saliva samples were collected from ten healthy individuals with no active carious lesions or history of antibiotics in the last three months. The participants were instructed not to brush their teeth 24 h and not eat or drink two h before the collection. The collected saliva samples were mixed to create a homogenous and complex bacterial composition. The use of saliva as inoculum was approved by the University of Maryland Baltimore Institutional Review Board (HP-00050407). The collected saliva was mixed with glycerol (70:30) and stored at −80° C. This model was used in previous studies to form thick and mature multispecies biofilm (24-26).
Dentin slabs (6×6×1 mm) were used as substrates to grow the biofilm (
To inoculate the saliva-derived biofilm model, the saliva-glycerol stock was mixed with McBain artificial saliva (1:50) and placed inside the wells of a 24-well plate containing the dentin slabs. The components of the McBain growth medium were as the following: mucin (Type II, porcine, gastric), 2.5 g/L; bacteriological peptone, 2.0 g/L; tryptone, 2.0 g/L; yeast extract, 1.0 g/L; NaCl, 0.35 g/L, KCl, 0.2 g/L; CaCl2, 0.2 g/L; 50 mM pipes, 15 g/L; hemin, 0.001 g/L; vitamin K1, 0.0002 g/L, at pH 7, and 0.2% of sucrose (28 81). Each dentin slab was immersed with 1.5 mL of the inoculum. The growth medium was replaced after 8 and 24 h, and then daily till seven days of biofilm growth. Later, the dentin slabs were subjected to antimicrobial photodynamic therapy treatment via 100 μg/mL of TBO, 2.5% MagTBO, 2.5% MagTBO with the magnetic field, and control with no treatment. The slabs were transferred to a vial containing 1 mL CPW solution, and the biofilms were harvested by vertexing and sonication. The following agar plates were prepared to enumerate the grown species:
a. Tryptic soy blood agar to count the total microorganisms.
b. Mitis salivarius agar (MSA) supplemented with potassium tellurite to count the total streptococci.
c. MSA supplemented with potassium tellurite and 0.2 units of bacitracin per mL to count mutans streptococci.
d. Rogosa agar to count the total lactobacilli.
The bacterial suspensions were serially diluted and plated in the agar plates and incubated for 48 h at 37° C. in 5% CO2, except for the rogosa plates where the incubation was continued for 4 to 5-day.
After biofilm collection, the Vicker's microhardness of dentin slabs was assessed as described (27). The amount of microhardness reduction was measured in percentages (%) as
The saliva-derived biofilm was initiated over dentin slabs, as described in the previous section. The dentin slabs were subjected to antimicrobial photodynamic therapy treatment via 100 μg/mL of TBO, 2.5% MagTBO, 2.5% MagTBO with the magnetic field, and control with no treatment. Then, the dentin slabs were prepared for confocal microscopy and SEM as explained with the S. mutans biofilms.
Shapiro-Wilk test was used to evaluate the data normality and distribution. Then, one-way ANOVA was used to evaluate the cytotoxicity of the microemulsions, and two-way ANOVA and Tukey's post hoc tests were used to analyze the effect of the magnetic nanoparticles and the effect of the magnetic field against the biofilms. All tests were conducted using the statistical software package Sigma Plot 12.0 (SYSTAT, Chicago, Ill., USA), and the statistical significance was set at p<0.05.
MagTBO microemulsions were synthesized using a high-ultrasonication method (18,19). TBO (100 μg/mL; #T3260, Sigma-Aldrich, St. Louis, Mo.) and different mass fractions of Fe2O3 nanoparticles (0.25, 0.5, 1, 1.5, 2, 2.5, and 5 wt %) were added to a mixture of distilled water, eucalyptus oil (Spectrum, New Brunswick, N.J.), polysorbate 20 (AmericanBio, Canton, Mass.), and glycerol (
When some superparamagnetic iron(II) oxide nanoparticles exist around one SPION, the superparamagnetic superparamagnetic iron(II) oxide nanoparticles show cooperative magnetophoretic behaviors under the experimental conditions. The permanent magnet and multiple superparamagnetic iron(II) oxide nanoparticles will produce low magnetic gradients, too, close to that of individual SPION (7.6 T/m). The local magnetic gradient is much lower than 100 T/m. According to the magnetophoretic literature, cooperative magnetophoresis would occur and SPIONs would aggregate (29,30). The aggregating superparamagnetic iron(II) oxide nanoparticles would move faster than individual superparamagnetic iron(II) oxide nanoparticles discussed above.
Encapsulating the TBO inside the microemulsion was also associated with improved TBO's biocompatibility. A previous report showed that 100 μg/mL TBO did not induce significant toxicity against mouse monocyte-macrophage cells (12). However, the results of this study illustrated that TBO alone was very toxic against the human gingival fibroblasts, as the percentage of fibroblast viability was 33.4%. Furthermore, the unloaded microemulsion was associated with a high cytotoxicity against the cells. Accordingly, the cytotoxicity of the microemulsion components was examined. Eucalyptus oil and polysorbate 20 were associated with significant cytotoxicity when added to the fibroblast media in a ratio similar to their proportion in the designed microemulsion. Previous investigations reported the eucalyptus oil's capability to disrupt bacterial membrane (31,32). Therefore, it is highly possible that the contact between eucalyptus oil and gingival fibroblasts cells negatively affected the viability of the cells. However, the cytotoxicity effect of polysorbate 20 against the cells was unexpected. Polysorbate 20 is well-known for being safe and biocompatible (33), and has high safety when injected intramuscularly in animals (34). The polysorbate 20 viscosity, rather than its cytotoxicity, interfered with the cell attachment during the incubation, resulting in less viable cells.
When the TBO was incorporated inside the microemulsion, the viability increased by 1-fold, resulting in more enhanced viability. Such results may encourage higher concentrations of TBO within the microemulsions to maximize the antibacterial reduction, as increasing the concentration of TBO is associated with an enhanced antibiofilm effect (12). Unlike most of the nanoparticles, increasing the superparamagnetic iron(II) oxide nanoparticles concentration was associated with improved biocompatibility. The possible explanation of such findings may be attributed to the capability of superparamagnetic iron(II) oxide nanoparticles in conducting an intrinsic peroxidase-like activity (35,36). Therefore, the cell growth promotion induced by superparamagnetic iron(II) oxide nanoparticles could be achieved by reducing the intracellular H2O2, which may oppose the cytotoxic effect of TBO, eucalyptus oil, and polysorbate 20 (36). Besides, several investigations have shown the positive impact of iron in the cell cycle progression (37,38). It can be concluded that using the microemulsion as a drug carries and the conjugation of superparamagnetic iron(II) oxide nanoparticles reduced the cytotoxicity of the other components within the microemulsion.
Following the fibroblast cells viability assay, three microemulsions were selected for further investigations; 1, 2.5, and 5% MagTBO microemulsions. Table 1 shows that all the microemulsions passed the stress tests as no phase separation was observed. The pH of the microemulsion was very low, indicating a highly acidic solution. Using the spectrophotometer to monitor the absorbance changes, minor differences were observed at 3 and 6 months. The long-term evaluation showed that all the microemulsions were stable for up to six months, whereafter the microemulsions started to show some significant physical changes. The density of the synthesized microemulsions was slightly increased compared to TBO.
A variety of nanomaterials have been widely investigated to improve the stability and efficiency of photosensitizers for antimicrobial photodynamic therapy. These approaches involve the use of polymeric nanoparticles as a carrier or functionalizing the photosensitizer into metallic nanoparticles (39). TBO functionalized into chitosan improved the antibacterial action against P. gingivalis, Aggrebacter actinomycetemcomitans, and E. faecalis (40,41). TBO conjugated with several nanoparticles such as gold and silver had demonstrated a more significant antibacterial action compared to the use of TBO alone (42,43). Advanced investigations in antimicrobial photodynamic therapy have also focused on designing and optimizing systems that maximize the photosensitizers' antibacterial effect and improve their stability and biocompatibility, such as nanospheres and emulsions (15).
In the present invention, the TBO photosensitizer was functionalized into superparamagnetic iron(II) oxide nanoparticles inside microemulsions. Microemulsions are metastable colloidal systems with a wide range of applications in medicine and pharmacy (44) and have been used extensively to carry therapeutic agents. Microemulsions are composed of aqueous and organic phases that are dispersed in each other and stabilized by surfactants (emulsifiers) to control the surface tension between the two phases with a polar head and nonpolar tail at the oil-water interface (45). Microemulsions' droplet size ranges from 10 to 100 nm, with a higher thermodynamic stability than nano and macroemulsions, indicating that microemulsions are less likely to experience physical changes over time (46,33).
The designed microemulsions improve the TBO's biocompatibility, as was demonstrated above. Besides biocompatibility, several advantages can be obtained from using microemulsions as a drug delivery approach. Due to their small droplet size, microemulsions provide a good surface area to volume ratio concerning the drug's absorption, thus improving the bioavailability of the loaded drug (47). Besides, droplet size at a small scale increases the resistance of microemulsion against physical changes, resulting in better stability than other emulsions (48). Microemulsions can improve the solubility of poorly water-soluble substances and enhance the biocompatibility of the loaded materials (49,50). In the present invention, the synthesized microemulsions demonstrated good thermodynamic and long-term stabilities responding to different stress challenges and improved the biocompatibility of the TBO photosensitizer.
Antibiofilm Efficacy of MagTBO Platform into the Photodynamic Process
Representative agar plates for the control and different treatment protocols are shown in
Reactive oxygen species (ROS) and free radicals in the living cells are kept in balance as they are very destructive when produced in high quantities (51). Photosensitizers with no light activation cannot generate reactive oxygen species, allowing antimicrobial photodynamic therapy to have high specificity for aiming the targeted organs or tissues, one of the main advantages of antimicrobial photodynamic therapy (52). When photosensitizers at a specific wavelength are activated, a high amount of reactive oxygen species is induced. It absorbs the hydrogen found in the cell wall of the targeted cells, which minimizes the cells' wall leading to lysis due to the turgor pressure (53). The use of aPDT has gained much attention recently due to its ability to eradicate bacterial biofilms without inducing bacterial resistance (54). It has been hypothesized that the photo-oxidative damage caused by antimicrobial photodynamic therapy is very aggressive against bacterial biofilms. Thus, the surviving microorganisms are very weak in initiating adaptive mechanisms toward aPDT (55).
In dentistry, antimicrobial photodynamic therapy has been extensively investigated to control several oral diseases, including caries, periodontitis, peri-implantitis, and endodontic infections (10). As a photosensitizer, TBO is a member of the nonporphyrin phenothiazinium family capable of targeting the bacterial membrane and accumulating inside the mitochondria to attack the targeted cells (12). TBO has shown a great antibacterial effect against oral pathogens such as S. mutans, Enterococcus faecalis, and Porphyromonas gingivalis (15). TBO at the concentration of 100 μg/mL was used herein. This concentration did not induce significant toxicity against the macrophage cell line, while concentrations higher than 100 μg/mL were reported with critical cytotoxicity (12).
The clinical outcomes concerning using aPDT in vivo have shown limited effectiveness of less than 2-log reduction (11,13,14). Several photosensitizers' drawbacks may contribute to that, mainly the hydrophobicity of photosensitizers that may limit their penetration through biofilms (56). Besides, photosensitizers are highly susceptible to aggregation when mixed with aqueous solution and prone to degradation due to light sensitivity and elongated storage (57,58). As a result, applying nanotechnology to functionalize photosensitizers may help eliminate these obstacles in antimicrobial photodynamic therapy. The design of nanosystems overcomes the photosensitizers' drawbacks, improves their activity, and enhances biocompatibility (50,59). Most of the reported studies concerning microemulsions aimed to improve the drug delivery to target cancer cells (33). On the contrary, few investigations were conducted to enhance the antimicrobial photodynamic therapy outcomes by incorporating photosensitizers into microemulsions to target pathogenic microorganisms, such as Candida albicans, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa (60,61).
To robust the antibacterial activities of the TBO-microemulsion, SPIONs were incorporated. Using magnetic nanoparticles is guided by applying a magnetic field force to achieve site-directed drug delivery (62). The most commonly used magnetic nanoparticles in the literature are iron oxide nanoparticles (63). Multiple preclinical and clinical studies have shown the capability of magnetic nanoparticles and magnetic fields in improving the delivery of anticancer drugs to devastate different types of malignant tumors (64).
Magnetic nanoparticles and magnetic fields may induce destructive heat against the targeted cells, inducing apoptosis and cell death (65,66). Moreover, magnetic nanoparticles can internalize inside the targeted cells, allowing the combined therapeutic agents to precisely exert the need for intercellular activity (67). It is contemplated that the magnetic force can direct the particles to biofilms' core, resulting in severe physical damage to the bacterial colonies (68,69).
The effect of the microemulsion containing the superparamagnetic iron oxide nanoparticles without the photosensitizer was examined under the magnetic field force (
In antimicrobial photodynamic therapy targeting oral biofilms, encapsulating chlorine e6 (Ce6) and coumarin 6 (C6) with iron oxide magnetic nanoparticles was associated with significant inhibition of 4- to 5-log against periodontal pathogens (70). The present invention combined the microemulsion approach and superparamagnetic iron oxide nanoparticles to improve the antimicrobial photodynamic therapy performance. However, in
Eucalyptus oil was used herein as an organic phase, polysorbate 20 and glycerol as surfactants. Several investigations found that eucalyptus oil has an antimicrobial effect by disrupting microorganisms' cell membranes and allowing the conjugated antibacterial agents to maximize their effect (20,31,32). However, the use of eucalyptus oil alone did not reduce the S. mutans biofilm in this report (
On the basis of these observations, a saliva-derived multispecies biofilm was prepared to investigate the effectiveness of the aPDT via the MagTBO microemulsion against thick and mature multispecies biofilms. Saliva was used as an inoculum to grow the multispecies biofilms and establish a more challenging situation for the MagTBO microemulsion. The use of human saliva as inoculum was demonstrated in several studies to provide more clinically relevant biofilms (80,24,25). The saliva mixture collected from 10 individuals can provide a homogeneous bacterial community and allow for analyzing the growth of different bacterial species. Mutans streptococci and lactobacilli were investigated as they are among the leading pathogens in coronal and root caries (24). Different studies reported that clinically isolated microorganisms might not recover the same quantity and homogeneity when grown in vitro, as several species could be lost (80,26). Therefore, such a model cannot be relied on to resemble the oral cavity's microbiota exactly. The same issue is applied when discussing the selective agar media used to isolate and enumerate the bacterial species (81).
The following references are cited herein.
This non-provisional patent application claims benefit of priority under 35 U.S.C. § 119(e) of provisional application U.S. Ser. No. 63/270,193, filed Oct. 21, 2021, the entirety of which is hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63270193 | Oct 2021 | US |
