الفهرس 
general introduction
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Abstract
Optimization of localized colloidal systems for the treatment of periodontal pockets
Summary
Periodontal diseases are a group of inflammatory conditions affecting the supportive structures of the teeth, the gingiva, the periodontal ligament and the alveolar bone. They are characterized by destruction of the periodontal ligament, resorbtion of the alveolar bone and the migration of the junctional epithelium along the tooth surface with the formation of a space (pocket) between the gum and the teeth and can eventually cause tooth loss. This pocket provides an ideal environment for the growth and proliferation of microbes including anaerobic pathogenic bacteria.
As specific bacteria are thought to play a major role in disease process, antimicrobial agents have been used as adjuncts to mechanical treatment. The potential side-effects in administering systemic antibiotics and the inability of antiseptic mouthwashes to penetrate the periodontal pocket have fuelled interest in the localized delivery of therapeutic agents within the periodontal pocket, thus ensuring a high effective concentration of antimicrobial agents at the site of infection.
Local drug delivery systems that are commercially available as controlled release devices suffer from several potential problems, including insufficient spectrum of antimicrobial activity against some periodontal polymicrobial infections, risks of producing an antibiotic resistant microbiota, and high acquisition costs.
In this context, combination therapy of antimicrobials in a local drug delivery system will be of great impact. Metronidazole is very effective against anaerobic bacteria which predominates in periodontal diseases. However, it is not effective against aerobic organisms, and has only marginal coverage for some of the anaerobic gram-positive organisms. Therefore, metronidazole should always be administered with an agent effective against aerobic or facultative anaerobes. Doxycycline is a semi-synthetic tetracycline derivative, which is active against a wide spectrum of bacteria. Thus it was desired to formulate colloidal carrier systems containing doxycycline hydrochloride and metronidazole that could be used in the treatment of periodontitis by direct periodontal intrapocket administration. To fulfill this target, the work in this thesis was divided into three chapters:
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Chapter I: Preparation and characterization of doxycycline hydrochloride and metronidazole loaded proniosomal gel
This chapter included the preparation and characterization of proniosomes containing doxycycline hydrochloride (DH) and metronidazole (MT). Slurry method using maltodextrin as carrier was used to prepare proniosomes consisting of different types of spans (span 20, span 40, span 60 and span 80) alone and in combination with cholesterol (CH) as membrane stabilizer. The niosomal suspensions obtained after proniosomes hydration were evaluated in terms of photomicroscopy, transmission electron microscopy, percent drug entrapment, particle size, zeta potential and in-vitro release. The influence of different formulation variables such as surfactant chain length, CH content, drug amount and surfactant loading per gram carrier on the entrapment efficiency, particle size, zeta potential and in-vitro release rate of both drugs from different preparation was investigated.
Proniosomes characterization was carried out through scanning electron microscopy, powder flowability, differential scanning calorimetry and FT-IR spectroscopy. Stability studies were carried out by investigating the leakage of both drugs from the prepared proniosomes when stored at ambient and refrigeration temperatures for three months. The optimized proniosomes powder was incorporated in poloxamer gel to allow easy application into the periodontal pocket.
from the work in this chapter the following could be concluded
1- Maltodextrin was a good carrier for the preparation of proniosomes due to its smooth surface and good flow properties.
2- Proniosomes powder spontaneously formed niosomal suspensions upon hydration with buffer at temperature above the surfactant transition temperature.
3- Niosomes derived proniosomes prepared from span 60 and span 40 gave the highest entrapment efficiencies of DH and MT, followed by span 20 and span 80. This was attributed to the longer chain length and higher transition temperature of span 40 and span 60.
4- The effect of CH incorporation on drugs entrapment efficiencies varied according to the non-ionic surfactant used. A significant increase in entrapment efficiencies of DH and MT were obtained when 10 mole% of CH was incorporated into niosomes prepared from span 40 and span 60 followed by a decrease in
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encapsulation efficiencies of the drugs upon further increase in CH content. CH increases the rigidity of the bilayer and lowers the permeability of the drugs resulting in greater drug retention up to an optimum level (10 mole%) after which it starts to disrupt the bilayer leading to decreasing drugs entrapment. However, CH was found to have little effect on both drugs entrapments into span 20 and span 80 based niosomes.
5- Increasing the surfactant loading from 1 to 3mM/gram carrier resulted in a significant decrease in the entrapment efficiencies for both drugs. Further increase of surfactant loading to 5mM/gram carrier showed non-significant decrease in entrapment efficiencies as compared to formula prepared at 3mM/gram carrier. Increasing the surfactant loading resulted in a decrease in the number of formed niosomes because of the inefficient hydration of the proniosomes
6- For most niosomal suspensions prepared from different types of spans, DH showed a slightly higher entrapment than MT. This was attributed to the different structure of the two drugs.
7- As the drug amount increased from 10 to 40mg, the % entrapment efficiencies of both drugs were increased. However, further increase of drug amount to 60mg resulted in a significant decrease in % entrapment efficiencies of both drugs.
8- The mean vesicle size of niosomal suspensions formed at 1mM/gram carrier was in the range of 383-2073nm. Niosomal suspensions prepared from span 20 and span 40 formed smaller vesicles than those prepared from span 60 and span 80. Also, niosomal suspensions prepared using span 20 and span 40 showed higher zeta potential than span 60 niosomes. Increasing the CH content increased the vesicles size, however zeta potential values were not affected upon varying the CH content.
9- Vesicles obtained at low surfactant loading were smaller than those obtained at high surfactant loading with a non-significant difference in zeta potential values.
10- Vesicles obtained at low drug amount were smaller than those obtained at higher drug amount, however, the zeta potential value was not affected upon varying the drug amount.
11- The in-vitro release of DH and MT from different niosomal suspensions showed an initial burst effect of the contained drugs, which varied depending on the
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composition of the formulations. This was followed by a slower release rate of the remained entrapped drug up to 8 hours. Niosomal suspensions prepared from span 40 and span 60 showed a slower release rate than those prepared from span 20 and span 80.
12- CH incorporation reduced the percent of DH and MT released from niosomal suspensions. Proniosomes formed from span 60 and CH in molar ratio 9:1 showed the slowest rate of release for both drugs.
13- Increasing the surfactant loading from 1 to 3 and 5mM/gram carrier resulted in a significant increase in the release rate of both drugs.
14- SEM images of proniosomes prepared at the lowest surfactant loading (1 mM/gram carrier) appeared very similar to the SEM images of the maltodextrin carrier with more uniform, smooth and thin coating. As the surfactant loading was increased to 3 or 5 mM/gram, the surface became quite rough with thick and uneven coat.
15- The results of the flowability of maltodextrin and proniosomes prepared at different surfactant loadings were consistent with the SEM images in which the angle of repose of 1 mM/gram surfactant loading proniosomes powder was smaller than that of pure maltodextrin indicating better flowability, however increasing the surfactant loading increased the thickness of the coat and the roughness of the particles which resulted in poorer flowability.
16- The thermotropic behavior of DH and MT in proniosomes powder revealed the transformation of the native crystalline forms of the drugs to amorphous state, indicating their efficient encapsulation.
17- The FT-IR spectra of pure DH, MT, maltodextrin, CH, span 60, their physical mixture and loaded proniosomes revealed no chemical interaction between both drugs and the components of the prepared proniosomes.
18- Proniosomes as a powder offered a satisfactory stability when stored in refrigerator for 3 months. Drugs leakage was lower at refrigeration temperature than at ambient temperature.
19- Optimized proniosomes formula formed of span 60 and CH in molar ratio 9:1 with 40 mg of each drug and 1mM lipid/ gram carrier was incorporated into poloxamer gel. The in-vitro release results showed that the release of both drugs
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from proniosomal gel was sustained to 24 hours and fitted Higuchi release kinetics.
Chapter II: Preparation and characterization of doxycycline hydrochloride and metronidazole loaded solid lipid microparticles gel
This chapter included the preparation and characterization of solid lipid microparticles containing DH and MT. Hot homogenization method was used to prepare solid lipid microparticles (SLMs) using different types of lipids and stabilized using different types and concentrations of surfactants. The influence of different formulation variables such as lipid type (tripalmitin, tristearin, Compritol and stearic acid), homogenization speed, total drug amount, varying DH to MT percent, different types and concentrations of surfactant(s), addition of glyceryl monostearate to lipid matrix on the entrapment efficiency, particle size and zeta potential was investigated. The in-vitro release study was performed on selected formulations. Freeze drying of the optimized SLMs was performed without and with the addition of two types of cryoprotectants (mannitol, trehalose) in different concentrations (5,10,15%w/w). Characterization of freeze dried SLMs included scanning electron microscopy, differential scanning calorimetry, FT-IR spectroscopy, in-vitro release and stability study.
from the work in this chapter the following could be concluded
1- SLMs prepared from tristearin and tripalmitin showed the highest entrapment efficiencies of DH and MT followed by Compritol and stearic acid.
2- Decreasing the homogenization speed from 8000 to 4000 rpm resulted in an increase in the entrapment efficiencies of DH and MT, which may be attributed to the larger particle size formed at lower speed, thus accommodating more drugs.
3- The entrapment efficiency was dependent on the drug amount, increasing drugs amount increased the entrapment efficiencies of both drugs till a certain limit, after which increasing the drugs amounts resulted in a significant decrease in the entrapment efficiencies. The two drugs differed in the amount needed to achieve maximum entrapment efficiency which may be attributed to the difference in their
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partition coefficient. A defined amount of DH (25mg) was needed to achieve maximum entrapment efficiency, and further increase in this amount resulted in a decrease in entrapment efficiency, in contrast a greater amount of MT (100mg) was added to achieve maximum entrapment in the prepared SLMs due to its greater affinity for the lipid, without affecting DH entrapment efficiency.
4- The entrapment efficiencies of DH and MT in SLMs differed according to type and concentration of surfactant used. The combination of two surfactants resulted in a significant increase in the entrapment efficiencies of both drugs into SLMs in comparison to those stabilized using one surfactant. Increasing the concentration of surfactant(s) from 2.5% to 5%w/w resulted in a decrease in the entrapment efficiencies of DH and MT.
5- The incorporation of glyceryl monostearate with tripalmitin resulted in a significant decrease in the entrapment efficiencies of DH and MT.
6- The mean particle size of SLMs formed of Compritol was larger than those formed from tripalmitin, tristearin and stearic acid. This may be attributed to Compritol high melting point which increases the melt viscosity and decreases the homogenization efficiency. SLMs prepared from different lipids showed negative zeta potential values.
7- Homogenization at higher rpm (8000) resulted in smaller SLMs compared to homogenization at lower rpm (4000) which resulted in larger particles. There was no significant difference in the zeta potential values at both speeds.
8- Increasing the concentration of the two drugs resulted in a non-significant increase in the size of the formed microparticles.
9- The in-vitro release of all tested SLMs showed an initial burst effect of the contained drugs, which varied according to the composition of the formulation. This was followed by a slower continuous release of the remained entrapped drugs for 48 hours and the kinetics of drugs release fitted Peppas release kinetics.
10- SLMs formulae formed of tripalmitin lipid matrix at 4000 rpm with 25mg DH and 100mg MT and stabilized using 5%w/w Epikuron or 5%w/w poloxamer 188+Epikuron showed the highest entrapment efficiencies and slowest rate of release of both drugs among the other tested SLMs formulations and therefore were chosen to be subjected to freeze drying.
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11- SLMs stabilized using two surfactants (5%w/w poloxamer 188+Epikuron) showed higher entrapment efficiency after freeze drying than SLMs stabilized using one surfactant (5%w/w Epikuron).
12- All tried cryoprotectants did not result in significant improvement in the entrapment efficiencies of both drugs. SLMs prepared without cryoprotectant and with 5%w/w mannitol showed the highest entrapment efficiencies of DH and MT with non-significant difference between them, therefore both were selected for further evaluation.
13- The release rate of DH and MT from freeze dried SLMs did not differ significantly from that of SLMs before freeze drying.
14- SEM images of freeze dried SLMs prepared using 5%w/w tripalmitin and stabilized using 5%w/w poloxamer 188+Epikuron without cryoprotectant appeared very similar to the SEM images of freeze dried SLMs with 5%w/w mannitol.
15- The thermotropic behavior of DH and MT in freeze dried SLMs powder revealed the transformation of the native crystalline forms of the drugs to amorphous state, indicating their efficient encapsulation.
16- The FT-IR spectra of pure DH, MT, TP, P188, their physical mixture and loaded SLMs revealed no chemical interaction between both drugs and the components of the prepared SLMs.
17- Freeze dried SLMs offered a satisfactory stability when stored at ambient and refrigeration temperatures for three months.
18- Optimized formula of freeze dried SLMs formed of tripalmitin lipid matrix at 4000 rpm with 25mg DH and 100mg MT and stabilized using 5%w/w poloxamer 188+Epikuron was incorporated in poloxamer gel to allow easy application. The in-vitro release results showed that the release of both drugs from SLMs gel was sustained for 72 hours and fitted Peppas release kinetics.
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Chapter III: Clinical and microbiological evaluation of the selected formulae
In this chapter the efficacy of the selected proniosomes and SLMs formulations incorporated into poloxamer gel was evaluated clinically in comparison to scaling and root planning (SRP) alone and to SRP in addition to medicated poloxamer gel on selected patients suffering from periodontitis. Histopathological evaluation was performed to ensure the safety of the formulations after application on mice gingival tissue. The study was carried on twelve systemically healthy patients suffering from periodontitis over two weeks period. All the selected patients had at least four pockets with probing pocket depth of 4-7 mm. The patients were divided into four groups, group 1 (SRP) was treated by SRP alone, group 2 (SRP+G) was treated by both SRP and poloxamer gel, group 3 (SRP+P) was treated by SRP and proniosomal gel and finally group 4 (SRP+S) was treated by SRP and solid lipid microparticles gel. Topical application of gels was performed after scaling and at day 7 after the start of the treatment. Follow-up was done on basis of changes in both bacteriological counts (total anaerobic and aerobic counts) and clinical parameters. The investigated clinical parameters were plaque index (PI), gingival index (GI), papillary bleeding index (PBI), probing pocket depth (PPD) and clinical attachment level (CAL). Bacteriological counts were done at baseline, after scaling, at days 3, 7 and 14 after the start of treatment, while clinical parameters were measured at baseline and at the end of the study.
from the work in this chapter the following could be concluded
1- Histopathological evaluation of proniosomal gel and SLMs gel revealed the safety of the formulations on the gingival tissue without causing any inflammation or tissue reaction
2- Group 2 (SRP+G) showed a non-significance difference in the anaerobic count throughout the study and in the percentage decrease in the total anaerobes after 2 weeks from group 1 (SRP). The observed results indicated the inability of the poloxamer gel to further decrease the anaerobic count after scaling, which may be due to its rapid leakage and washing from the pocket. The significance decrease in the anaerobic count in group 3 (SRP+P) and group 4 (SRP+S) in comparison to
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group 1 (SRP) throughout the study indicates the additional effect of the proniosomal and SLMs gels in decreasing the bacterial count which is attributed to their ability to sustain the release of the two drugs.
3- A non-significant change was observed in the aerobic bacterial count over time and between the different treatments which may be due to the nature of the counted organisms (pathogenic organisms or normal flora) under aerobic conditions. The decrease in the pathogenic species may be accompanied by an increase in the normal flora of the mouth (e.g cocci) resulting in a non-significant change in the total count.
4- All treatments in the different groups resulted in a significant improvement in the measured clinical parameters (PI and GI) from baseline to the end of the study.
5- All other clinical parameters (PBI, PPD and CAL) showed a significant improvement from baseline to 2 weeks, with higher improvement in group 3 (SRP + P) and group 4 (SRP + S).
6- Based on the results, the adjunctive use of either proniosomal gel or SLMs gel to conventional mechanical debridement resulted in an improvement in microbiological and clinical parameters in chronic periodontitis patients.
7- Further longer clinical studies on periodontitis patients are needed to investigate the effect of the local combined therapy on the clinical parameters over longer time.
8- Proniosomes and SLMs gels would offer additional benefit as adjunctive to SRP for the treatment of periodontitis.