Formulation and Evaluation
of Antibacterial Creams and Gels Containing Metal Ions for Topical Application
Abstract
Background. Skin
infections occur commonly and often present therapeutic challenges to
practitioners due to the growing concerns regarding multidrug-resistant
bacterial, viral, and fungal strains. The antimicrobial properties of zinc
sulfate and copper sulfate are well known and have been investigated for many
years. However, the synergistic activity between these two metal ions as
antimicrobial ingredients has not been evaluated in topical formulations.
Objective. The aims of the present study were to (1) formulate topical creams
and gels containing zinc and copper alone or in combination and (2) evaluate
the in vitro antibacterial activity of these metal ions in the formulations.
Method. Formulation of the gels and creams was followed by evaluating their
organoleptic characteristics, physicochemical properties, and in vitro
antibacterial activity against Escherichia coli and Staphylococcus aureus.
Results. Zinc sulfate and copper sulfate had a strong synergistic antibacterial
activity in the creams and gels. The minimum effective concentration was found
to be 3 w/w% for both active ingredients against the two tested microorganisms.
Conclusions. This study evaluated and confirmed the synergistic in vitro
antibacterial effect of copper sulfate and zinc sulfate in a cream and two
gels.
1.
Introduction
Topical skin
infections commonly occur and often present therapeutic challenges to
practitioners, despite the numerous existing antimicrobial agents available
today. The necessity for developing new antimicrobial means has increased
significantly due to growing concerns regarding multidrug-resistant bacterial,
viral, and fungal strains [1–4]. Consequently, attention has been devoted to
safe, new, and/or alternative antimicrobial materials in the field of
antimicrobial chemotherapy.
Common examples for
topical skin infections include diaper rash, cold sores, and tinea (also called
pityriasis) versicolor. Diaper rash is a
form of irritant contact dermatitis. It is one of the most common
dermatological conditions encountered in babies while using diapers [5] and is
estimated to occur in 7–35% of babies between the ages of 9 and 12 months [6].
Its development is multifactorial, including skin wetness, friction, skin
irritants, and pH change, which favors the growth of microorganisms including
Candida, Staphylococcus, and Streptococcus [7]. It has been shown that zinc and
copper ions have antimicrobial activity against Staphylococcus aureus and
Candida albicans [8]. Cold sores (also known as herpes labialis) are a common
viral infection occurring on the lips, primarily caused by herpes simplex virus
(HSV) type 1 [9]. Studies have shown that zinc and copper salts exhibit
inactivation of HSV both in vivo and in vitro [10–13]. Zinc sulfate was found
to have an antimicrobial effect in treating cold sores [14]. The molecular
mechanism of its therapeutic effect was found to be the drastic inactivation of
free virus in skin tissues, intercellular vesicles, and blisters [15].
Pityriasis versicolor is a superficial fungal infection of the skin, usually
caused by Malassezia species. It is one of the most common skin diseases in
tropical and subtropical areas and is characterized by fine scaly patches and
macules [16]. Both zinc sulfate and copper sulfate have been found to be
effective in treating this disease [17, 18].
In recent years, a
number of metal ions have been studied as potential antimicrobial agents,
including silver [19], copper [20], zinc [21], iron [22], magnesium [23], and
titanium [24]. Zinc, alone or as an adjuvant, has been found to be advantageous
in a number of dermatological infections and inflammatory diseases owing to its
modulating actions on macrophage and neutrophil functions, natural killer
cell/phagocytic activity, and various inflammatory cytokines. Zinc sulfate has
been studied in vivo in a number of diseases, including warts [25], herpes
genitalis [26], pityriasis versicolor [18], and acne vulgaris [27] in varying
concentrations. Copper is well known for its antimicrobial properties. It has
been used as an algicide, germicide, and fungicide for decades. Several
antimicrobial mechanisms of copper were proposed in recent articles, including
reactive hydroxyl radical formation leading to damaged cell integrity,
denaturation of DNA by binding of copper to protein molecules, and inactivation
of enzymes and obstruction of functional groups of proteins from displacement
of essential ions [28–30]. Additionally, topically applied copper sulfate and
hypericum perforatum were found to be efficacious in vivo in the treatment of
herpes skin lesions [13].
The antimicrobial
activity of zinc sulfate and copper sulfate has been investigated for many
years. However, the synergistic activity between these two metal ions as
antimicrobial ingredients has not been evaluated in topical formulations. The
aim of the present study was to formulate topical creams and gels containing
zinc sulfate or copper sulfate, and a combination of these, and to evaluate the
in vitro antibacterial activity of these metal salts in the formulations
against Escherichia coli and Staphylococcus aureus. The in vitro antibacterial
activity of the formulated products was also compared to commercial products
available for the treatment of diaper rash and cold sores.
Incorporating metal
ions such as zinc and copper often creates a formulation challenge due to the
high reactivity of these ions. Even trace amounts of metal ions are able to
catalyze oxidation reactions in fatty compounds in products, leading to
deterioration including odor formation, color change, and physical and/or
chemical instability [31]. Metal ion reactions with the ingredients in the
formulations can affect the quality, efficacy, consumer appeal, and shelf-life
of formulations. Stability of product and of the antibacterial activity was
studied for 12 weeks at two different temperatures in two different containers.
2. Materials and
Methods
2.1. Materials
Copper sulfate
pentahydrate was purchased from Fagron, Inc. (St. Paul, MN). Zinc sulfate
heptahydrate, Carbomer 940, refined corn oil, almond oil sweet, lecithin soya
granular, glycerin, and propylene glycol were purchased from Letco Medical
(Decatur, AL). (ι)-Carrageenan was purchased from Sigma-Aldrich (St. Louis,
MO). Hypromellose (Benecel, K4M PHARM, also known as hydroxypropylmethyl
cellulose, HPMC), and Prolipid 141 (a mixture of glyceryl stearate, behenyl
alcohol, palmitic acid, stearic acid, lecithin, lauryl alcohol, myristyl
alcohol, and cetyl alcohol) were received as gifts from Ashland (Wilmington,
DE). Kollidon® 90F (poly vinylpyrrolidone, PVP) was obtained from BASF
(Ludwigshafen, Germany). Poloxamer 407 was purchased from PCCA (Houston, TX).
FlexiThix™ (2-pyrrolidinone-1-ethenyl homopolymer) was received as a free
sample from ISP Technologies, Inc. (Wayne, NJ). Xanthan gum, guar gum,
methylparaben, propylparaben, butylated hydroxytoluene (BHT), and citric acid
monohydrate were obtained from Spectrum Chemical (Gardena, CA). Medium chain
triglycerides (MCT) were obtained from Mead Johnson & Company (Evansville,
Indiana). Soybean oil, Cithrol™ GMS 40 (glyceryl stearate), Arlacel™ 165 (a
mixture of glyceryl stearate and PEG-100 stearate), Tween 60, and Span 80 were received
as free samples from Croda, Inc. (Edison, NJ). PEG-16 Macadamia and PEG-10
Sunflower were obtained from FloraTech (Gilbert, Arizona). Cocoa butter was a
gift from Koster Keunen, Inc. (Watertown, CT). Cetyl alcohol, stearic acid,
stearyl alcohol, and isopropyl myristate were obtained from Sherman Research
Labs (Toledo, OH). Coconut oil was purchased from Spectrum Organic Products,
(Melville, NY). Tefose HC (a mixture of cetyl alcohol, glyceryl stearate,
ceteth-20, and steareth-20) was a free sample from Gattefossé (Saint-Priest
Cedex, France). PEG-8 beeswax was a gift from Koster Keunen, Inc. (Watertown,
CT). Urea was purchased from Gallipot®, Inc. (St. Paul, MN). Triethanolamine
was purchased from Making Cosmetics (Snoqualmie, WA). Mueller-Hinton agar and
gentamicin 10 μg standard discs were purchased from Becton, Dickinson and
Company (Sparks, MD). The marketed products included Equate® Diaper Rash Relief
Cream (distributed by Walmart, Inc.), Nexcare™ Cold Sore Treatment Cream
(distributed by 3M), and Campho-Phenique® Cold Sore Treatment Gel (distributed
by Bayer Health Care LLC), which were all purchased at a local Walmart store
(Toledo, OH). All ingredients used in the various formulations can be found in
Tables 1 and 2.
Table 1: Composition
of the topical cream formulations.
Table 2: Composition
of the topical gel formulations.
2.2. Methods
2.2.1. Formulation of
the Topical Cream
The oil phase was
prepared by melting the waxes at 75°C and mixing the ingredients uniformly. The
aqueous phase was prepared by dissolving the water-soluble ingredients in
deionized water. The water phase was warmed to 75–80°C until all ingredients
were dissolved. When the water and oil phase were at the same temperature, the
aqueous phase was slowly added to the oil phase with moderate agitation and was
kept stirred until the temperature dropped to 40°C. The emulsion was cooled to
room temperature to form a semisolid cream base. Zinc sulfate and copper
sulfate were dissolved in warmed deionized water, and the solutions were added
to the cream base using an overhead stirrer (Talboys Engineering Corp, Emerson,
NJ). The mixture was stirred for 15 min until the formulation became uniform.
The drug-loaded cream was preserved with paraben concentrate. The exact
concentration of each ingredient is shown in Table 1.
2.2.2. Formulation of
the Topical Gels
When using
(ι)-carrageenan, xanthan gum, and guar gum, the powder polymers were dispersed
in 75°C warm deionized water with stirring. When all the polymers were
dissolved, the mixture was removed from the hot plate. The desired amount of
zinc sulfate and copper sulfate was dissolved in the clear gel with intensive
stirring. The mixture was then cooled to room temperature and preserved with
paraben concentrate.
In formulations where
HPMC was the thickening agent, the polymer was dispersed in 75°C warm deionized
water with stirring. The resulting solution was stored at room temperature
overnight until a clear gel formed. Zinc sulfate crystals and then copper
sulfate crystals, after complete dissolution, were dispersed into the gel with
intensive agitation. Preservative was added to the formulation in the last
step.
Poloxamer was
dissolved in cold water and stored under refrigerated conditions at 4°C for a
night. The oil phase was prepared by mixing lecithin and isopropyl myristate in
a 1 : 1 ratio. The mixture was stored at room temperature overnight for the
complete dissolution of lecithin. The active ingredients were then added
directly to the aqueous phase. The gel was prepared by mixing 1 part of oil
phase with 4 parts of aqueous phase (poloxamer gel) using a vortex mixer
(VORTEX-T, Genie® 2, Bohemia, NY).
Kollidon 90F,
FlexiThix, and Carbomer 940 were directly dispersed into deionized water at
room temperature with intensive agitation. Active ingredients were incorporated
into the gel uniformly. In order for Carbomer 940 to form a gel,
triethanolamine was added to neutralize the pH to 6–6.5. Table 2 shows the
amount of ingredients used for the gels.
2.2.3. Physical
Evaluation of the Topical Formulations
(1) Organoleptic
Characteristics. All blank formulations (i.e., formulations without any active
ingredients or preservatives) and drug-loaded formulations were tested for
physical appearance, color, texture, phase separation, and homogeneity. These
characteristics were evaluated by visual observation. Homogeneity and texture
were tested by pressing a small quantity of the formulated cream and gels
between the thumb and index finger. The consistency of the formulations and
presence of coarse particles were used to evaluate the texture and homogeneity
of the formulations. Immediate skin feel (including stiffness, grittiness, and
greasiness) was also evaluated.
(2) Spreadability.
Spreadability of the formulations was determined by measuring the spreading
diameter of 1 g of sample between two horizontal glass plates (10 cm × 20 cm)
after one minute. The standard weight applied to the upper plate was 25 g. Each
formulation was tested three times.
(3) pH Values. One
gram of each formulation (including the blank, i.e., formulation without any
active ingredients or preservatives, and drug-loaded formulation) was dispersed
in 25 mL of deionized water, and the pH was determined using a pH meter
(Mettler-Toledo Ingold Inc., Billerica, MA). Measurements were made in
triplicate. The pH meter was calibrated with standard buffer solutions (pH 4,
7, and 10) before each use.
(4) Viscosity
Measurement. A Brookfield viscometer DV-I (Brookfield Engineering Laboratories,
Middleboro, MA) was used with a concentric cylinder spindle #29 to determine
the viscosity of the different topical formulations. The tests were carried out
at 21°C. The spindle was rotated at 0, 0.5, 1, 2, 2.5, 4, 5, 10, 20, 50, and
100 rpm values. All measurements were made in triplicate.
2.2.4. In Vitro
Antibacterial Activity
(1) Preparation of
Mueller-Hinton (MH) Agar Plates. Mueller-Hinton (MH) agar medium was prepared
according to the manufacturer’s instructions and autoclaved for 20 minutes at
20 psi. After autoclaving, the agar medium was cooled to 40–45°C in a water
bath. Sixty mL of the cooled agar medium was poured onto the prepared mm petri
dish. The agar was allowed to cool to room temperature and stored in a
refrigerator (2–8°C) until used.
(2) Preparation of
Inoculum. Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29213)
were used to evaluate the antibacterial activity of the topical formulations
containing zinc sulfate and copper sulfate. The microorganisms were subcultured
the previous day to ensure that the tested microorganisms were in their log
phase of growth and to ensure the validity of the results. One or two isolated
colonies of the tested microorganisms were touched using a sterile cotton swab.
The microorganisms were suspended in 2 mL of sterile saline medium and vortexed
well until a uniform suspension was obtained. The turbidity of the suspension
was measured at 625 nm using a UV-Vis spectrophotometer (Thermo Scientific,
Waltham, MA). The turbidity of the suspension was adjusted to a 0.5 McFarland
standard by adding more microorganism if the suspension was too light or
diluting with sterile saline if the suspension was too heavy. The suspension
was prepared before inoculating the microorganisms on the agar plate.
(3) Inoculation of
the MH Plate. To inoculate the MH agar plates, a sterile cotton swab was dipped
into the suspension and streaked over the surface of the agar plates. This
procedure was repeated three times; each time, the plate was rotated
approximately 60 degrees to ensure even distribution of the inoculum [32]. The
plates were then allowed to dry at room temperature for 5 min before applying
the drug.
(4) Preparation of
Agar Well Diffusion Assay. The dried inoculated MH agar plates prepared above
were used to perform the agar well diffusion assay. A sterile cork borer was
used to make the wells by punching holes on the inoculated MH agar plates. Each
well was 5 mm in diameter, and the cut-out of the agar was removed using a
sterile needle. A desired amount of the formulations was weighed and placed
into each well on an analytical balance. Gentamicin 10 μg standard discs were
used as a control to ensure that the agar medium was appropriate to support the
growth of the microorganism beyond the zone of inhibition. The gentamicin
standard disc was placed and pressed gently onto the same inoculated agar plate
by using sterile forceps. The inoculated agar plate was incubated at 37°C for
18 hours. The observed diameters of the zones of inhibition were measured by
using a ruler to the nearest millimeter.
First, in order to
observe how effective the active ingredients were alone versus combined, 20 μL
of 3% copper sulfate, 3% zinc sulfate, 6% copper sulfate, 6% zinc sulfate, and
% copper sulfate and zinc sulfate combined solutions were tested for antibacterial
activity against E. coli and S. aureus. Next, the selected cream and gel
formulations containing both active ingredients in a series of concentrations
(including 0, 0.1, 0.25, 0.5, 1, 2, and 3% of each ingredient) were tested for
antibacterial activity against E. coli and S. aureus to evaluate their
effective concentration. Gentamicin 10 μg standard disc was used as the
control. The sample size directly measured into the wells was μg in this
study. Finally, the selected cream and gel formulations were directly compared
to the marketed products, including Nexcare Cold Sore Treatment Cream,
Campho-Phenique Cold Sore Treatment Gel, and Equate Diaper Rash Relief Cream,
in terms of their antibacterial activity against E. coli and S. aureus. The
sample size directly measured into the wells was μg in this study.
2.2.5. Stability
Study
The antibacterial
activity of all selected drug-loaded formulations was tested against E. coli
using the above-described agar well diffusion assay for 12 weeks (measurements
were made on day 1, week 3, week 6, week 9, and week 12). The antibacterial
activity of the formulations was compared for samples stored at room
temperature (25°C) and in the refrigerator (4°C) as well as those packaged into
glass containers versus plastic containers. In addition to the antibacterial
activity, pH values, color, physical appearance, and texture were also tested
during the 12 weeks with the above-described methods.
2.2.6. Statistical
Analysis
Statistical analysis
of data was performed using one-way ANOVA (Tukey’s post hoc test). A difference
was considered statistically significant when .
3. Results
From the twenty
different creams formulated, C1 was selected as the final formulation for
further testing. Eighteen different gels were formulated in this study, from
which G1 and G5 were selected as optimal formulations for further evaluation.
3.1. Physical
Evaluation of Topical Cream and Gels
3.1.1. Organoleptic
Characteristics
The organoleptic
properties, including physical appearance, color, texture, phase separation,
homogeneity, and immediate skin feel of the selected topical formulations, are
displayed in Table 3. Results showed that the cream and both gels had a
cosmetically appealing appearance and smooth texture, and they were all
homogenous with no signs of phase separation. All formulations were blue due to
copper sulfate.
Table 3:
Physicochemical evaluation of selected topical formulations.
3.1.2. Spreadability
Spreadability of
semisolid formulations, that is, the ability of a cream or gel to evenly spread
on the skin, plays an important role in the administration of a standard dose
of a medicated formulation to the skin and the efficacy of a topical therapy.
Figure 1 shows the spreading values, that is, diameters observed for the
formulations, after one minute. The values refer to the extent to which the
formulations readily spread on the application surface by applying a small
amount of shear. Results indicated that our cream and gels had comparable
spreadability to that of commercial products used as comparators in the study.
Figure 1:
Spreadability values for the selected cream (C1) and gels (G1 and G5) compared
to various marketed products (, results shown as mean ± SD).
3.1.3. pH Values
The pH values for the
blank and drug-loaded cream and gels are shown in Table 4. The pH of the
formulations decreased when the active ingredients were added to the bases. The
pH of the skin normally ranges from 4 to 6. The pH of the cream was more acidic
than that of the skin, while the gels’ pH values were similar to the skin’s
normal pH value. The pH values of the formulations did not change significantly
over the period of 12 weeks.
Table 4: pH of blank
and drug-loaded formulations at day 1 and at week 12.
3.1.4. Viscosity
Measurement
Viscosity values for
the drug-loaded cream and gels are shown in Figure 2. All products had a
pseudoplastic behavior, as expected. C1 and G1 had a similar viscosity curve,
while G5 had a lower initial viscosity.
Figure 2: Viscosity
curves for the selected cream (C1) and gels (G1 and G5) (, results shown as
mean ± SD).
3.1.5. In Vitro
Antibacterial Activity
The in vitro
antibacterial study was performed by measuring and comparing the diameter of
zones of inhibition (in mm) for the various products. The zone of inhibition
can be defined as the clear region around the well that contains an
antimicrobial agent. It is known that the larger the zone of inhibition, the
more potent the antimicrobial agent.
In the first step,
the two active ingredients’ antibacterial activity was measured individually
and combined against E. coli and S. aureus. It can be concluded from the
results that the antibacterial activity of zinc sulfate was higher than that of
copper sulfate against the tested microorganisms (Figure 3). Results also
confirmed that copper sulfate and zinc sulfate have a synergistic activity, as
shown by their larger zone of inhibition against tested microorganisms ().
Figure 3:
Antimicrobial activity of copper sulfate and zinc sulfate solutions in various
concentrations against Escherichia coli and Staphylococcus aureus (, results
shown as mean ± SD) ().
In the next step, the
antibacterial activity of the selected cream (C1) and gels (G1 and G5) with
varying amounts of active ingredients (0, 0.1, 0.25, 0.5, 1, 2, and 3%) was
studied against E. coli and S. aureus. The results are shown in Table 5 and
Figure 4. The blank formulation did not contain any active ingredients or any
preservative. The formulation named paraben contained preservative but no active
ingredients. Gentamicin 10 μg standard disc was used as the control in the
study. No zone of inhibition was observed for the blank, paraben, and 0.1%
strength formulations for either the cream or the gels. The zones of inhibition
increased as the concentration of copper sulfate and zinc sulfate increased.
This indicated that the antibacterial activity of copper sulfate and zinc
sulfate increased against E. coli and S. aureus as the concentration of the
actives was increased. As for the C1 formulation, the antibacterial activity of
the sample containing 2% active ingredients was as good as that of the control
(gentamicin) against S. aureus, while the antibacterial activity of the sample
containing 3% active ingredients was statistically significantly higher than
the control and the rest of the samples against both E. coli and S. aureus ().
A visual representation of these results for C1 can be found in Figure 4. In
the case of G1, similar results were seen. Samples containing 2% active
ingredients had a similar antibacterial activity to that of the control, while
the samples containing 3% active ingredients had statistically significantly
higher activity against both microorganisms. As for G5, both the 2% and 3%
samples had a similar antibacterial activity as the control against E. coli,
while only the samples containing 3% of each active ingredient had an
antibacterial activity comparable to the control against S. aureus. Based on
the results, it can be concluded that both active ingredients have to be present
in a concentration of at least 3% to achieve a similar or better antibacterial
activity as the control against E. coli and S. aureus.
Table 5:
Antimicrobial activity of the selected gel formulations in various dilutions
against Escherichia coli and Staphylococcus aureus ().
Figure 4:
Antimicrobial activity of the selected cream (C1) containing copper sulfate and
zinc sulfate in various concentrations against (a) Escherichia coli and (b)
Staphylococcus aureus (, results are shown as mean ± SD) ().
The final in vitro
antimicrobial study was performed to compare the antibacterial activity of the
selected formulations to those of marketed products against E. coli and S.
aureus. As there is currently no marketed product available with zinc sulfate
and copper sulfate, two commercially available cold sore gels and a diaper rash
cream, Nexcare Cold Sore Treatment Cream, Campho-Phenique Cold Sore Treatment
Gel, and Equate Diaper Rash Relief Cream, were used as comparators in the
study. Nexcare Cold Sore Treatment Cream contains the following active
ingredients: benzocaine as an external analgesic and allantoin as a skin
protectant. The active ingredients in Campho-Phenique Cold Sore Treatment Gel
are camphor and phenol as pain relievers or antiseptics, and, in Equate Diaper
Rash Relief Cream, zinc oxide is a skin protectant. The results of this study
are displayed in Figure 5. Results indicated that the antibacterial activity of
C1, G1, and G5 was similar to that of the control (gentamicin), while the
marketed products had a significantly lower activity against the two tested
microorganisms. G1 and G5 had higher antibacterial activity against the tested
bacteria than C1, which may be related to the composition of these products.
The cream formulation contained oily components, which are immiscible with
water and may slow down the diffusion of the drugs from the cream base.
Figure 5:
Antimicrobial activity of the selected cream (C1), gels (G1 and G5), and three
marketed products (Nexcare Cold Sore Treatment Cream, Campho-Phenique Cold Sore
Treatment Gel, and Equate Diaper Rash Relief Cream) against Escherichia coli
and Staphylococcus aureus (, results shown as mean ± SD).
3.2. Stability Study
All formulations
maintained their blue color and intensity of color for 12 weeks in all storage
conditions. Similarly, the physical appearance, homogeneity, and texture of all
formulations remained the same by the end of the storage period. None of the
formulations showed signs of physical or chemical instability in any of the containers
or at any of the temperatures. The antibacterial activity of all formulations
was maintained for 12 weeks in both containers and at both temperatures.
Results are shown in Figure 6 for G5 as well as in Table 6 for C1 and G1.
Table 6: Antimicrobial
activity during the stability study.
Figure 6:
Antimicrobial activity of one of the selected gels (G5) during the stability
testing compared to the control (gentamicin) against Escherichia coli (,
results shown as mean ± SD).
4. Discussion
Twenty different
cream bases (C1–C20) were formulated using different ingredients in varying
concentrations. After incorporating the active ingredients into the bases, the
physical stability of a number of bases was affected negatively by the metal
ions, leading to creaming and breaking of the emulsions. Formulations C5–C20
suffered from such issues; therefore, they were discontinued from further
characterization. Formulations C1–C4 were formulated with the same ingredients,
but with varied concentrations of the emulsifiers and thickeners. These four
creams had similar consistency, and no apparent change in their physical
appearance was observed after adding the metal salts. Based on the overall
evaluation for physical appearance and immediate skin feel, C1 was selected as
the final formulation for further testing.
In addition to the
creams, eighteen different gels (G1–G18) were formulated using eight different
gelling agents. Only two polymers, namely, carrageenan and HPMC, proved to be
optimal gelling agents for the metallic active ingredients used. G1 and G2 were
formulated using carrageenan. These gels incorporated the active ingredients
well. They had similar texture, consistency, and viscosity. The appearance,
viscosity, and skin feel provided by G1 was considered better for a topical
gel; therefore, it was selected for further testing. G3–G6 were formulated
using different concentrations of HPMC. Out of these four samples, only G5
provided an optimal gel. To be considered optimal in this study, a gel (1) had
to be homogenous without showing signs of physical or chemical instability; (2)
had to be able to dissolve and keep the active ingredients in a dissolved form
without precipitating or aggregating; and (3) had to have a high enough
viscosity not to flow off the skin during/after application. In the case of G3
and G4, precipitation or aggregation of the polymers was observed when the
active ingredients were added to the 5% HPMC gel. The explanation for this is
that the amount of water used in these gels was not enough to dissolve the
crystalline actives and keep the gelling agent dispersed. G4 contained more
water compared to G3; however, even that higher amount did not prove to be
enough for a stable formulation. G5 met all our requirements; therefore this
formulation was selected for further testing. G6 had a too low viscosity, which
was not deemed appropriate for a topical gel. As for the other polymers used,
including xanthan gum, guar gum, poloxamer 407, Kollidon 90F, FlexiThix, and
Carbomer 940, the gels’ physical stability was compromised when the active
ingredients were added to the gel bases.
The three selected
formulations were optimal in terms of their appearance, homogeneity, and
viscosity. Previous studies [6–9] indicated that both active ingredients had
antibacterial activity, which this study confirmed. The two metal salts had a
synergistic activity when combined in the creams and gels, which was also
confirmed by our study. The second antibacterial study indicated that both zinc
sulfate and copper sulfate have to be present in at least a 3% concentration in
order to achieve similar or better results against the tested microorganisms
than gentamicin, which was used as the control. As there are no marketed
products available today with copper sulfate and/or zinc sulfate, commercial
products for the treatment/prevention of cold sores and diaper rash with active
ingredients other than copper sulfate and zinc sulfate were used as comparators
in the final antibacterial study. This limited study showed that the formulated
creams and gels had a significantly higher antibacterial activity against the
two tested microorganisms than the marketed products. With a properly planned
and conducted follow-up study using additional microorganisms—preferably fungi,
bacteria, and viruses—the true antimicrobial activity of the products could be
evaluated, and a more realistic comparison could be made.
The stability study
indicated that both gels and the cream were able to maintain their integrity
for 12 weeks without showing signs of instability. Additionally, the main
function, that is, antibacterial activity of all formulations, remained stable
for the tested time period, which is promising. As discussed in the
introduction, incorporating metal salts into emulsions and gels can be a
challenging task for formulators due to the high reactivity of these salts. Many
of our formulations were affected by the metal ions, which resulted in
precipitation, phase separation, color change, and other forms of instability.
Only a few formulations, that is, the selected creams and gels, were able to
remain stable after incorporating the active ingredients into them.
5. Conclusions
In this study,
various creams and gels were formulated with copper sulfate and zinc sulfate,
which act as antimicrobial agents. During the formulation process, the quality,
appearance, and stability of many creams and gels were affected by the highly
reactive metal ions. A cream and two gels were found to be optimal for our
purpose, and these were selected for further evaluation based on their physical
properties, in vitro antibacterial activity, and product stability. Although
only a small fraction of the formulated products were deemed optimal, a great
achievement is that the integrity, pH values, texture, appearance, and
antibacterial activity of these selected products were maintained for 12 weeks.
A major finding of
this study is that copper sulfate and zinc sulfate have a synergistic
antibacterial activity in creams and gels. The minimum effective concentration
in vitro was found to be 3% for both active ingredients. A properly planned and
conducted in vitro follow-up study could confirm the antimicrobial activity of
the formulations against other microorganisms, and a more realistic comparison
could be made between our products and marketed products for various skin
conditions.
