An In Vitro Study to Determine the Physicochemical, Mechanical, and Antibacterial Properties of a Novel Spirulina Containing Controlled Release Intrapocket Drug Delivery System.

CONTEXT: Periodontitis is primarily an inflammatory condition caused by an array of microorganisms present in dental plaque. Elimination or adequate suppression of periodontal pathogens in the subgingival microflora is essential for adequate periodontal healing to take place. The development of subgingivally placed controlled delivery systems has provided the possibility of effective intrapocket concentration levels of antibacterial agents for an extended period of time, resulting in an altered subgingival flora and enhanced healing of the attachment apparatus. Although a number of synthetic antimicrobial agents are being used as local drug delivery, currently, pharmaceutical technology development has focused on the ingredients derived from nature. Natural phytochemicals have proven to be worthy substitutes of their synthetic and chemical-laden counterparts owing to their extensive natural activity, advanced safety margins, and inferior costs so that they can be of huge benefits, especially to the lower socioeconomic population around the world and spirulina platensis (SP) is one such emerging remedy. AIMS: The aim of the study was to develop three controlled release drug delivery systems containing different concentrations of SP to be used inside the periodontal pockets. The study also aimed to determine the antimicrobial activity of all the three concentrations of SP drug delivery system against major periodontopathic microorganisms and to test the physicochemical properties of the delivery system that exhibited maximum antimicrobial efficacy so that the suitability of its use inside the periodontal pocket could be determined. SETTINGS AND
DESIGN: The study was an in vitro experimental design. SUBJECTS AND METHODS: Three different controlled release SP hydrogels (4%, 6%, and 12%) to be used inside the periodontal pockets were developed and antibacterial properties against periodontal pathogens were assessed. The hydrogel exhibiting maximum antimicrobial efficacy was then tested of physicochemical and mechanical properties to determine its suitability of its use inside the periodontal pocket. STATISTICAL ANALYSIS USED: Data were analyzed using one-way analysis of variance. Post hoc Tukey honestly significant difference test was used for comparison within the group and between the different groups.
RESULTS: 12% SP hydrogel was found to have maximum antimicrobial efficacy against major periodontal pathogens, and its physicochemical and mechanical properties were also optimum to be used inside the periodontal pocket.
CONCLUSIONS: 12% SP hydrogel can act as a promising adjunct to periodontal mechanical therapy and may also reduce the chances of more invasive periodontal surgical procedures. Copyright:

INTRODUCTION

Oral cavity is a home to about 500 different types of microorganisms that have been associated with a number of oral diseases and periodontitis is one such disease which is the leading cause of tooth loss in adults.[1] Periodontitis is primarily an inflammatory condition caused by an array of microorganisms present in dental plaque. Several suspected pathogens have been identified to be involved in this destructive periodontal disease. Some of the most important species are Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, and Prevotella intermedia.[2] Elimination or adequate suppression of periodontal pathogens in the subgingival microflora is essential for adequate periodontal healing to take place.[3] Current options for periodontal therapy include mechanical debridement of the root surfaces, oral hygiene measures, use of systemic and local chemotherapeutic agents, and periodontal surgical procedures.

Numerous studies have revealed that mechanical debridement alone cannot effectively eliminate some types of periodontopathogens, making it difficult to obtain or reestablish periodontal health.[4] Systemic antibiotics can serve as an adjunct to conventional mechanical therapy as they have the potential to penetrate the periodontal tissues and pocket through the serum and suppress the tissue invading periodontal pathogenic bacteria that could potentially cause re-infection.[5] However, administration of systemic antibiotics is not without adverse effects, the most important being the emergence of multiple drug resistance in the population. Moreover, with the use of systemic antibiotics, local concentration of drugs fails to reach the minimum inhibitory concentration for pathogens, thereby the growth of these pathogens is ineffectively controlled.[6] Surgical intervention is beneficial but cannot be performed in medically compromised cases and in patients unwilling to go for invasive surgical procedures.[7] Local delivery of antibiotics can be used to overcome these limitations. Local drug delivery (LDD) can achieve the bioavailability to surrounding tissues as it can reach the base of the periodontal pocket and maintained for an adequate amount of time for the antimicrobial effect to occur.[8]

Although a number of synthetic antimicrobial agents are being used as LDD, currently, pharmaceutical technology development has focused on the ingredients derived from nature as they have minimal side effects, are economical, and equally effective when compared to their synthetic and chemical-laden counterparts.[9] Phytotherapy has been widely practiced in India for ages which are backed by appropriate scientific evidence.[10] Many of the medicinal plants are being used to treat systemic conditions such as diabetes mellitus, obesity, hypercholesterolemia, anemia, and atherosclerosis.[11] Spirulina platensis (SP) is one such emerging remedy that has been considered as safe by the US Food and Drug Administration (FDA).[12] It is a cyanobacterium or blue–green alga that has attracted much attention among the researchers due to its wide range of nutritional and health benefits. The micronutrients present in it such as carbohydrates, proteins, essential fatty acids, vitamins, magnesium, selenium, copper, manganese, zinc, and iron make it a highly beneficial nutraceutical agent.[13]

A vast number of studies have reported the antioxidant properties of phycocyanin, a major pigment present in SP, which has been used to explain spirulina's anti-inflammatory properties.[91415] There is also evidence from studies regarding its antimicrobial properties against organisms such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pyogenes, Vibrio cholerae, Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, Klebsiella pneumonia, and many more.[16] As far as available literature is concerned, none of the studies seemed to have explored the antibacterial properties of spirulina against periodontopathic microorganisms.

LDD methods that employ the use of mouth wash, gel, toothpaste, and supra and subgingival irrigation fail to maintain antimicrobial concentration in the subgingival area and also require high initial concentration and multiple applications to provide sustained effectiveness. To circumvent these drawbacks, controlled release devices which can directly target the microbes proliferating in the pockets have been developed. The desirable features for intrapocket localized drug delivery are mucoadhesivity, biodegradability, biocompatibility, nontoxicity, and protecting the active therapeutic molecule from premature degradation. Controlled release devices are available in the form of hydrogels, emulgels, films, tablets, fibers, and patches.[17]

To the best of our knowledge about the available literature, the present study was the first one that made an attempt to develop three controlled release drug delivery systems containing different concentrations of SP to be used inside the periodontal pockets. The study also aimed to determine the antimicrobial activity of all the three concentrations of SP drug delivery system against major periodontopathic microorganisms and to test the physicochemical properties of the delivery system that exhibited maximum antimicrobial efficacy so that the suitability of its use inside the periodontal pocket could be determined.

SUBJECTS AND METHODS

The present study was conducted in an in vitro experimental design. Three controlled release drug delivery devices in the form of hydrogel containing SP were prepared. The method of preparation described by Aslani et al.[18] was followed with slight modifications.

Source of algal extract

Spray-dried and standard quality of SP powder was obtained from Pondicherry Spirulina Farms, Pondicherry, India, for all experiments. SP powder was the active ingredient of the hydrogel.

Source of ingredients of base gel

Sodium carboxymethyl cellulose (NaCMC), Carbopol 934P, and polyethylene glycol 400 (PEG 400) were obtained from Bangalore Fine Chemicals, Bengaluru, India. Methylparaben, propyl paraben, and triethanolmine were obtained from Sigma-Aldrich Chemie Gmbh (Steinheim, Germany).

Preparation of base gel

Accurately measured amount of Carbopol 934P was slowly dispersed in 40 ml of distilled water as the water was continuously stirred by a mechanical stirrer at 1200 rpm for 30 min. The slow dispersion was done for proper hydration of the polymer molecules and to avoid clumping of the molecules of carbopol. In a separate beaker, accurately measured quantity of NaCMC was added to 40 ml of distilled water at 50°C with constant stirring at 2000 rpm for 30 min. Methylparaben and propyl paraben as preservatives were added in 5 ml of PEG 400 (acts as solublizer) and stirred properly. After complete dispersion of the two gelling agents in water, they were then mixed together and the preservative solution was added to it with constant stirring. Finally, the volume was made up to 100 ml by adding remaining amount of distilled water, and the pH was adjusted to 6–7 by drop-wise addition of triethanolamine to obtain a clear homogenous gel of required consistency. The base gel was without the active ingredient, i.e., SP powder and acted as negative control in the experiment.

Preparation of different formulations of spirulina platensis hydrogel

4% spirulina platensis hydrogel

The procedure as described for the preparation of base gel was repeated with some additions. Accurately weighed quantity of 4 g of SP powder was mixed separately in 5 ml of PEG 400. Methylparaben and propyl paraben as preservatives were added in 5 ml of PEG 400 and stirred properly. The SP solution and the preservative solution were added to the base gel with constant stirring. Finally, the volume was made up to 100 ml by adding remaining amount of distilled water and the pH was adjusted to 6–7 by adding a few drops of triethanolamine to obtain a clear homogenous gel of required consistency.

6% spirulina platensis hydrogel

The procedure as described above was repeated except that accurately measured 6 g of SP powder was added to 5 ml of PEG 400.

12% spirulina platensis hydrogel

The above procedure as described above was repeated except that accurately measured 12 g of SP powder was added to 5 ml of PEG 400.

The entire composition of the base gel and different formulations of SP gel has been shown in Table 1. All the four formulations (three formulations of SP gel and base gel as negative control) were kept and sealed in sterile, amber colored glass bottles, and named as sample A, B, C, and D, respectively, and sent for microbiological analysis. Blinding was done to avoid bias.

Table 1

Composition of different concentrations of spirulina platensis hydrogel

Ingredients in grams	Different concentrations of SP hydrogel
	4% SP gel	6% SP gel	12% SP gel	Base gel*
Carbopol 934P (gelling agent)	1	1	1	1
Sodium CMC (gelling agent)	3	3	3	3
SP powder (active ingredient)	4	6	12	-
PEG 400 (solubilizer)	10	10	10	-
Methylparaben (preservative)	0.18	0.18	0.18	0.18
Propyl paraben (preservative)	0.02	0.02	0.02	0.02
Triethanolamine (pH balancer)	qs	qs	qs	qs
Distilled water qs to	100	100	100	100

*Base gel is without the active ingredient (SP powder) and act as negative control. SP: Spirulina platensis, CMC: Carboxymethyl cellulose, PEG: Polyethylene glycol

Antimicrobial testing

The antibacterial effect of different gel formulations against major periodontal pathogens, namely, A. actinomycetemcomitans, P. gingivalis, T. forsythia, Prevotella Intermedia, and F. nucleatum was determined using disc diffusion method. For disc diffusion method, the organisms were first grown in pure culture. The broth was prepared. The lawn of respective organisms was prepared on the plates. Wells were then cut on the plates, one each for different SP gels, positive control and negative control. In the present study, doxycycline was used as positive control (metronidazole as positive control for F. nucleatum and T. forsythia). Samples were then added in amount of 50 µl in each well. The plates were then incubated in anaerobic jar for 48 h. After 48 h, the plates were taken out and the diameter of zone of inhibition was measured using a vernier calliper to the nearest whole millimeters. The microbiological procedure was repeated in triplicates for each bacterium, and corresponding three values of zones of inhibition for different formulations of SP gel along with the two controls were obtained for each of the five bacteria. The values so obtained were compared within the group, between different groups (different formulations of SP gel), and also with positive control. The antibacterial activity was determined as low, moderate, and high when the zone of inhibition was 10–15 mm, 15–20 mm, and ≥20 mm, respectively.[19]

The formulation that exhibited maximum antibacterial activity was optimized and sent to laboratory for testing of its physicochemical properties to determine its suitability for use inside the periodontal pockets. The physicochemical properties were characterized as Aapearance and clarity, syringeability, pH, viscosity, % drug content (phycocyanin), gelation temperature, gelation time, mucoadhesive strength, and in vitro percentage drug release. Mechanical properties such as firmness, consistency, and cohesiveness of the formulated gel were evaluated with the help of a texture profile analyzer (TA-XT, Haslemere, Surrey, USA).

Statistical analysis

Statistical analysis was done using the software Statistical Package for theSocial Sciences (SPSS, version 21.0; SPSS Inc., Chicago, IL, USA). The values of zones of inhibition of different formulations of SP gel and control against five major periodontal pathogens were entered in the SPSS software for statistical analysis. Data were analyzed using one-way analysis of variance (ANOVA). Post hoc Tukey Honestly significant difference (HSD) test was used for comparison within the group and between the different groups. Statistical significance was established at P < 0.05.

RESULTS

Three different formulations of SP hydrogel were prepared by varying the concentrations of the active ingredient, i.e., SP powder to check the differences in the antimicrobial activity of the formulated gels against five major periodontal pathogens – A. actinomycetemcomitans, P. gingivalis, T. forsythia, P. intermedia, and F. nucleatum.

Zones of inhibition exhibited by SP gel at three different concentrations, positive control, and negative control against five major periodontal pathogens – A. actinomycetemcomitans, P. gingivalis, T. forsythia, P. intermedia, and F. nucleatum is shown in Table 2. The negative control was found to be inactive in antimicrobial activity and hence was not considered further for statistical analysis. The positive control displayed the widest zones of inhibition against all the five bacteria. SP gel showed increasing zones of inhibition against all the five pathogens as the concentration of the active ingredient in the SP gel increased. Mean zones of inhibition for each formulation (including positive control) and each bacterium were calculated for analysis [Graph 1]. The microorganism – T. forsythia was found to be weakly susceptible to all the three concentrations of SP gel, while P. intermedia displayed weak susceptibility to 4% and 6% SP gel and moderate susceptibility to 12% SP gel. A. actinomycetemcomitans, P. gingivalis, and F. nucleatum exhibited moderate susceptibility to 4% and 6% SP gel. However, 12% SP gel displayed wide zones of inhibition against A. actinomycetemcomitans, P. gingivalis, and F. nucleatum. A. actinomycetemcomitans, P. gingivalis, and P. intermedia showed susceptibility to doxycycline, while T. forsythia and F. nucleatum were susceptible to metronidazole as explained by the widest inhibition zones displayed [Table 2].

Table 2

Zones of inhibition (in mm)

Spirulina gel (%)	Microorganism tested
	Aa			Pg			Fn			Pi			Tf
4	15	15	14	15	15	16	14	15	15	13	12	12	12	13	12
6	18	15	17	17	18	18	16	18	18	14	14	13	13	13	14
12	21	22	24	24	22	24	23	22	22	15	14	16	13	12	15
Base gel (negative control)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Doxycycline* (positive control)	24	22	21	25	25	27	23	22	24	27	23	24	23	24	24

*Metronidazole as positive control in case of Fn and Tf. All values are in millimeters. Aa: Aggregatibacter actinomycetemcomitans, Pg: Porphyromonas gingivalis, Fn: Fusobacterium bacterium, Pi: Prevotella intermedia, Tf: Tanerella forsythia, Fn: Fusobacterium nucleatum

Graph 1

Comparison of mean inhibition zones against Aa, Pg, Tf, Pi, and Fn. Mean zones of inhibition by different concentrations of spirulina gel and positive control against Aa: Aggregatibacter actinomycetemcomitans, Pg: Porphyromonas gingivalis, Tf: Tannerella forsythia, Pi: Prevotella intermedia, Fn: Fusobacterium nuceatum

One-way ANOVA as shown in Table 3 revealed an increase in the mean zones of inhibition against all the five bacteria as the concentrations of active ingredient in the SP gel increased. It was statistically significant for all the bacteria except T. forsythia. Post hoc Tukey HSD test clearly revealed that 12% SP gel has a significant antimicrobial efficacy against A. actinomycetemcomitans, P. gingivalis, and F. nucleatum as compared to 4% and 6% SP gel formulation. However, for the microorganism P. intermedia, 12% SP gel showed a significant difference in antimicrobial activity when compared with 4% SP gel but not with 6% SP gel.

Table 3

Antimicrobial efficacy of different concentrations of spirulina gel (analysis of variance and post hoc-Tukey)

MO	Concentration of spirulina gel, mean±SD			P	Post hoc Tukey HSD test
	4%	6%	12%		T/t pairs	Tukey HSD P	Tukey HSD inference
Aa	14.67±0.5774	16.67±1.5275	22.33±1.5275	0.0009*	4% versus 6%	0.2191	Insignificant
					4% versus 12%	0.0010	P<0.01*
					6% versus 12%	0.0040	P<0.01*
Pg	15.33±0.5774	17.67±0.5774	23.33±1.1574	<0.0001*	4% versus 6%	0.0295	P<0.05*
					4% versus 12%	0.0010	P<0.01*
					6% versus 12%	0.0010	P<0.01*
Fn	14.67±0.5774	17.67±0.5774	23.33±1.1547	<0.0001*	4% versus 6%	0.0166	P<0.05*
					4% versus 12%	0.0010	P<0.01*
					6% versus 12%	0.0010	P<0.01*
Pi	12.33±0.5774	13.67±0.5774	15.00±1.0000	0.01*	4% versus 6%	0.1510	Insignificant
					4% versus 12%	0.0110	P<0.05*
					6% versus 12%	0.1510	Insignificant
Tf	12.33±0.5774	13.33±0.5774	13.33±1.5275	0.4219	4% versus 6%	0.4836	Insignificant
					4% versus 12%	0.4836	Insignificant
					6% versus 12%	0.8999	Insignificant

*P<0.05 is significant. MO: Micro-organism, Aa: Aggregatibacter actinomycetemcomitans, Pg: Porphyromonas gingivalis, Fn: Fusobacterium bacterium, Pi: Prevotella intermedia, Tf: Tanerella forsythia, SD: Standard deviation, T/t: Treatment, HSD: Honestly significant difference

When compared with the positive control, 12% SP gel showed equal antimicrobial activity against A. actinomycetemcomitans and nearly similar antimicrobial efficacy against P. gingivalis and F. nucleatum as displayed by the insignificant difference in the mean inhibition zones between the two in post hoc Tukey HSD test. However, for microorganisms P. intermedia and T. forsythia, the antimicrobial efficacy of the positive control clearly exceeded the efficacy of all the three SP gel formulations, as the widest zone of inhibition was exhibited by the positive control against the two respective microorganisms [Table 4].

Table 4

Comparison of antimicrobial efficacy of different concentrations of spirulina gel with positive control (analysis of variance and post hoc-Tukey honestly significant difference test)

MO	Concentration of spirulina gel, mean±SD				P	Post hoc-Tukey HSD test
	4%	6%	12%	Positive control†		T/t pairs	Tukey HSD P	Tukey HSD inference
Aa	14.67±0.5774	16.67±1.5275	22.33±1.5275	22.33±1.5275	0.0002*	4% versus 6%	0.3361	Insignificant
						4% versus 12%	0.0010	P<0.01*
						4% versus PC	0.0010	P<0.01*
						6% versus 12%	0.0010	P<0.01*
						6% versus PC	0.0010	P<0.01*
						12% versus PC	0.8999	Insignificant
Pg	15.33±0.5774	17.67±0.5774	23.33±1.1547	25.67±1.1547	<0.0001*	4% versus 6%	0.0553	Insignificant
						4% versus 12%	0.0010	P<0.01*
						4% versus PC	0.0010	P<0.01*
						6% versus 12%	0.0010	P<0.01*
						6% versus PC	0.0010	P<0.01*
						12% versus PC	0.0553	Insignificant
Fn	14.67±0.5774	17.33±1.1547	22.33±0.5774	23.00±1.0000	<0.0001*	4% versus 6%	0.0227	P<0.05*
						4% versus 12%	0.0010	P<0.01*
						4% versus PC	0.0010	P<0.01*
						6% versus 12%	0.0010	P<0.01*
						6% versus PC	0.0010	P<0.01*
						12% versus PC	0.7654	Insignificant
Pi	12.33±0.5774	13.67±0.5774	15.0±1.00	24.67±2.0817	<0.0001*	4% versus 6%	0.5656	Insignificant
						4% versus 12%	0.1065	Insignificant
						4% versus PC	0.0010	P<0.01*
						6% versus 12%	0.5656	Insignificant
						6% versus PC	0.0010	P<0.01*
						12% versus PC	0.0010	P<0.01*
Tf	12.33±0.5774	13.33±0.5774	13.33±1.5275	23.67±0.5774	<0.0001*	4% versus 6%	0.5613	Insignificant
						4% versus 12%	0.5613	Insignificant
						4% versus PC	0.0010	P<0.01*
						6% versus 12%	0.8999	Insignificant
						6% versus PC	0.0010	P<0.01*
						12% versus PC	0.0010	P<0.01*

*P<0.05 is significant, †Metronidazole as positive control in case of Fn and Tf while tetracycline is positive control in case of Aa, Pg and Pi. MO: Micro-organism, Aa: Aggregatibacter actinomycetemcomitans, Pg: Porphyromonas gingivalis, Fn: Fusobacterium bacterium, Pi: Prevotella intermedia, Tf: Tanerella forsythia, SD: Standard deviation, T/t: Treatment, HSD: Honestly significant difference

Since 12% SP gel showed maximum antimicrobial efficacy against the periodontal pathogens, this gel formulation was optimized for the testing of its physicochemical and mechanical properties, the results of which determined the suitability of the gel for use inside the periodontal pockets. The physicochemical and mechanical properties of 12% SP gel have been summarized in Table 5.

Table 5

Physicochemical and mechanical properties of 12% spirulina platensis gel

Test parameters	Result
Appearance and clarity	Green and clear
Syringeability	Syringeable through 21 gauge needle
pH	6.65±0.15
Percentage drug content (phycocyanin)	97.67±1.15
Gelation temperature (°C)	38±0.32
Gelation time (s)	40±0.15
Viscosity (cps)
5 (°C)	388±54
25 (°C)	1551±35
Mucoadhesive strength (g)	30.24±1.52
Firmness (g)	42±0.577
Consistency (g/s)	98.40±3.02
Cohesiveness (g)	25.12±2.69

In vitro drug release studies showed that the phycocyanin present in the SP was released up to 5 days, in which about 40%–60% of drug was released within 6 h [Graph 2].

Graph 2

Percentage of drug release of 12% spirulina gel formulation at predetermined time interval

DISCUSSION

Mechanical debridement of root surfaces forms the basis of periodontal therapy. However, ample amount of literature have reported that conventional mechanical therapy fails to completely eradicate the periodontopathic bacteria from the subgingival areas, especially those inhabiting the inaccessible areas such as furcations, root grooves, concavities, and deeper areas of pockets.[4]

Systemic antimicrobials can provide additional benefits when used as an adjunct to scaling and root planing. However, failure to reach the gingival fluid concentrations higher than the minimal inhibitory concentrations against the suspected pathogen, emergence of bacterial resistance, superinfections, and systemic side effects[6] have paved the way for the use of LDD systems that deliver the agents at the target areas at sufficient concentrations with lesser side effects and better patient compliance.[8]

The use of herbal extracts in the family of LDD agents has covered a new dimension in the nonsurgical therapy. Natural phytochemicals have proven to be worthy substitutes to synthetic antimicrobials owing to extensive natural activity, advanced safety margin, and inferior costs so that it can especially be of huge benefit to the lower socioeconomic population around the world. A vast number of in vitro experiments and clinical trials have been done with aloe vera, propolis, tulsi, cinnamon, coriander, acacia, green tea, and many more in the treatment of various forms of periodontitis.[20]

SP, a cyanobacterium, has emerged as a promising superfood as it is a rich source of iron, proteins, Vitamin A, B complex, and K, essential fatty acids, lipids, carbohydrates, and carotenoids such as B-carotene, which has antioxidant properties. A large body of evidence is in favor that phycocyanin, a major pigment present in SP, is responsible for its antioxidant properties, which in turn accounts for SP's anti-inflammatory action.[13] Numerous studies have also documented the broad-spectrum of activity against many Gram-positive and Gram-negative microorganisms such as S. aureus, S. epidermidis, S. pyogenes, V. cholerae, E. coli, B. cereus, P. aeruginosa, K. pneumoniae, and many more.[16] However, very little research has been done so far regarding the antibacterial property of SP against periodontal pathogens. Studies done by Mahendra et al.[14] and Khatavkar et al.[15] also focused on the anti-inflammatory effects of the spirulina gel and capsules, respectively, which were closely connected to the antioxidant property of the spirulina. Hence, in the present study, we formulated three different concentrations of SP hydrogel and studied the antibacterial activity of the three gels against five major periodontal pathogens – A. actinomycetemcomitans, P. gingivalis, T. forsythia, F. nucleatum, and P. intermedia.

The different concentrations of SP hydrogel that we developed were 4%, 6%, and 12%. The results of the studies done by Mahendra et al., where 4% SP gel was used in patients with chronic periodontitis and reduction in pocket depth and gain in clinical attachment level was obtained due to the gel's anti-inflammatory properties.[14] Similarly, Rostiny et al. used 6% and 12% spirulina chitosan gel on postextraction socket of Cavia cobaya and reported that 12% spirulina gel was significantly able to accumulate collagen fibers for faster healing and better bone remodeling after tooth extraction.[9] Hence, we opted to formulate the same concentrations of spirulina gel. The only difference was in the use of the type of vehicle to formulate the gel. Mahendra et al. formulated the gel with gelatin being the gelling agent or the vehicle. However, researchers have addressed that gels composed of gelatin do not have the property of sustained release.[14] Chitosan, although a natural, biocompatible, mucoadhesive, and biodegradable gelling agent is still not approved by the U. S. FDA as a marketed product in drug delivery owing to its limited knowledge on the biological processes involved in material–cell interaction and poor knowledge of polymer characteristics. Moreover, chitosan rather being an inert agent in the gel formulation has shown to exhibit a bacteriostatic activity.[11] Our attempt was to specifically look for antibacterial property of SP against periodontal pathogens and the use of chitosan as the vehicle might have had provided with false-positive results. Hence, in the present study, we preferred to use carbopol 934P as the gelling agent that has been extensively used in the controlled drug release formulations. Carbopol 934P is a high molecular weight carboxyvinyl polymer that is specifically intended for oral and mucosal applications. It is physiologically and microbiologically inert, has excellent temperature stability, has the ability to remain localized at a specific site, and possess good mucoadhesive properties[21] – one of the major requirements of a controlled drug delivery system to be effectively used inside the periodontal pocket. The periodontal pocket is continuously bathed by the gingival crevicular fluid (GCF). The GCF tends to be one of the main factors that result in faster and short-lived release of the active drug as opposed to what is required by an effective intrapocket LDD system.[22] Hence, the use of Carbopol 934P as the gelling agent was an optimum choice to carry out the experiment. NaCMC was used to further increase the viscosity of the formulation and aid in slow release of the drug.[23] The formulated gel was basically an aqueous-based gel or a hydrogel that can imbibe large amounts of water or biological fluids, while remaining insoluble.[24]

The present study was the first one to perform an in vitro antibacterial assessment of the three concentrations of SP hydrogel against A. actinomycetemcomitans, P. gingivalis, T. forsythia, F. nucleatum, and P. intermedia. A. Actinomycetemcomitans is an early colonizer that is a community activist, a necessary partner of a pathogenic consortium that encourages the overgrowth of other periodontopathogens.[25] P. gingivalis serves as the secondary colonizer often adhering to primary colonizers such as P. intermedia interacts with other members of the host microbiota and is considered as one of the prime etiological agents in the progression of inflammatory events of Stage 2, 3, and 4 periodontitis along with T. forsythia.[26] F. nucleatum is one of the prime co-aggregator of the oral cavity that plays a key role in dental plaque formation. It also facilitates intracellular and intercellular invasion of several other microbial species.[27] The results showed that SP gel at a concentration of 12% can effectively inhibit the growth of A. actinomycetemcomitans, P. gingivalis, and F. nucleatum comparable to that of positive control doxycycline (metronidazole in case of F. nucleatum). P. intermedia is also a primary colonizer that has been frequently isolated from the periodontal lesions of patients with Stage 2, 3 as well as 4 periodontitis, necrotizing ulcerative gingivitis, and puberty-induced gingivitis and has been shown to be resistant to several antibiotics.[1] In the present study, however, 12% SP gel showed moderate antimicrobial activity against this microorganism. Although the susceptibility of P. intermedia to 12% SP gel was far less than the positive control, the gel could still be considered as a possible alternative to its synthetic counterparts. Various studies have postulated that the phycocyanin pigment present in SP has excellent antioxidant, anti-inflammatory, and immune enhancement function that can probably explain its activity against the periodontal pathogens discussed so far. Further research is warranted to clear the views on this aspect. Due to scarcity of literature in specifically assessing the antimicrobial efficacy of SP gel against the periodontal pathogens, we were unable to perform any comparative analysis of the results. We encourage other researchers to carry out further studies assessing the same.

12% SP gel showed maximum antimicrobial activity among the three formulated gels, hence it was optimized for testing its physicochemical properties. 12% SP gel had the pH of 6.65 that was closer to the pH of GCF. Hence, this formulation can be used in periodontal pocket without any potential irritation.[17] The gelation temperature was 38°C ± 0.32°C, which was more or less close to body temperature. Viscosity results demonstrated that 12% SP gel is less viscous at temperatures 5°C, while highly viscous at temperatures 25°C. Lower viscosity is desirable for the in situ gel to be syringeable and get easily injected into the periodontal pockets, and once inside the periodontal pocket, a higher viscosity is required so that the drug remains inside the pocket for a longer time.[17] Mucoadhesive strength was also found to be optimum for its use inside the periodontal pocket. Mechanical properties such as firmness, consistency, and cohesiveness were determined by texture profile analyzer. For better syringeability, the gel should possess less firmness and consistency value which was very well exhibited by the 12% SP gel. Moreover, the cohesiveness of the gel was high that would aid in the full structural recovery following gel application.[17] In vitro release studies revealed a biphasic pattern of drug (phycocyanin) release, i.e., initial burst release in the first 6 h (50%–60% drug release) followed by slow and sustained release till 5 days. For periodontal applications, both the phases are desirable as the initial burst effect can be useful to achieve the required minimal inhibitory concentration of the periodontopathogens, while sustained release is required to maintain the antibacterial efficacy of the gel over longer periods.[28]

Hence, the results of testing of antibacterial assessment, physicochemical, and mechanical properties of 12% SP gel revealed that the said gel at 12% concentration is a suitable candidate for use as a controlled release drug delivery system inside the periodontal pockets to eradicate the infectious component in addition to the inflammatory lesion of the periodontal lesions.

The in vitro experimental design is one of the limitations of the study. The clinical trial of the SP gel in patients with periodontitis is in progress, which would establish a clear insight about the implications of SP gel in periodontal disease management. Another limitation was that the present study determined the antibacterial efficacy against only five periodontal pathogens. The effect of SP gel on other pathogenic bacteria present as a biofilm and a variety of viruses implicated in periodontal disease progression could not be determined.

CONCLUSIONS

Within the limitations of the present study, it can be concluded that such product, if found effective in further in vivo research, may provide an additional advantage to known beneficial anti-inflammatory and antioxidant properties of spirulina gel. The gel is not only technically easy to make but also economical and would also reduce the need for systemic as well as local antibiotics in a variety of periodontal procedures, thereby acting as a better adjunct to periodontal mechanical therapy and also reducing the chances of more invasive periodontal surgical procedures.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.