In vitro release and in vivo study of quercetin-loaded alginate-kappa carrageenan pulmospheres

Abstract

Some antituberculosis drugs were reported to have adverse effects. The study investigates the use of quercetin pulmospheres as an alternative to traditional antituberculosis drugs. Formulated with alginate and kappa carrageenan as F1, F2, and F3 (1:1, 1:2, and 1:3), the pulmospheres were observed for the release and deposition in rat lungs. Results show a sustained release of 50.47% ±0.43%–58.37% ±0.57% in 10 h above minimum inhibitory concentration (MIC) against Mycobacterium tuberculosis and provided Higuchi kinetics model. Pulmospheres delivered quercetin to the lungs and showed a deposition with high concentrations. The slowest rate was occurred in pulmospheres with polymer ratio of 1:2. Formula F2 showed the most optimal results with the lowest rhodamine B concentration of 11.934 ± 2.751–12.364 ± 0.070 µg/g and 6.987 ± 1.931–8.685 ± 2.672 µg/g for left and right lung, respectively, which produced same MIC compare to F1 and F3. The study suggests further evaluation of effective doses for antituberculosis.

INTRODUCTION

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. This disease primarily infects the lungs but can also affect other organs.[1,2] Currently, active agents from natural sources have antibacterial activity against Mycobacterium tuberculosis and are efficacious as adjunctive therapies are needed for tuberculosis therapies.[3]

Quercetin, a polyphenol flavonoid found in various plants, has antibacterial properties against Mycobacterium tuberculosis and has been used as adjunctive therapy to reduce tuberculosis symptoms.[4,5,6,7,8] However, it faces challenges such as chemical instability, solubility in water, and low permeability.[9,10] To deliver Quercetin to the lungs through inhalation, it should have an aerodynamic diameter of 1–5 μm, called pulmosphere particles consisting of a polymer matrix and calcium chloride (CaCl₂) crosslinker.[11,12,13,14] These factors help ensure effective delivery and reduce the risk of adverse effects.

Pulmospheres are effective carriers for drug release, with factors such as polymer expansion, drug-polymer ratio, erosion, and distribution affecting drug release. Quercetin pulp is ideal for sustained drug release, but the dose remains above the minimum inhibitory concentration (MIC).[15] Release of ciprofloxacin from microsphere at pH 7.4 for 12 h was found at 54.77%–61.90%.[16] Deposition profiles for pulmonary tuberculosis treatment must be within the therapeutic range and reduce drug use frequency to improve patient compliance.[17,18] The novelty of this study is the optimized quercetin pulmospheres, which are able to release active agent and be an effective carrier, were produced, as demonstrated by the deposition of quercetin in the lungs, showing their potency in lung delivery.

MATERIALS AND METHODS

Materials

Quercetin; Sodium Alginate (Sigma-Aldrich Inc.), Carrageenan (Danisco-Cultor), and CaCl₂.2H₂O (Solvay Chemicals International) is pharmaceutical grade; Na₂HPO₄ pro analysis (Merck); KH₂PO₄ pro analysis (Merck); NaCl pro analysis (Merck); Maltodextrin (PT Bratachem); HCl pro analysis (Merck); NaOH pro analysis (Merck); Aquademineralized (PT. Brata Chem).

Methods

Formulation of quercetin-alginate-carrageenan pulmospheres

Formulation of quercetin-alginate-carragenan pulmospheres is prepared as the previous method.[19]

In vitro release study

Quercetin release test from pulmospheres was performed in Phosphate Buffer Saline pH 7.4 ± 0.05 using a thermoshaker at 37°C at 100 rpm. Samples were taken at intervals of as much as 5.0 mL. Absorbance was observed using a spectrophotometer at a maximum wavelength of 370 nm. The concentration of dissolved quercetin was calculated, and a release profile curve was created. The rate of release is calculated from the value of b (slope) of the curve equation y = bx + a.

Determination of release kinetics

Drug release kinetics is determined by several kinetics equations. The determination of R² value of linear regression equation obtained from each formula. If the value is R² close to one, drug release kinetics from microsphere follows kinetics of the regression equation of the kinetics model. The kinetics models used are Zero Order Kinetics, First Order, Higuchi Model, and Korsmeyer Peppas.

Drug deposition test

Experimental animals

Ethical approval was obtained from the research ethics committee, Veterinary Medicine Faculty, Airlangga University (No: 2.KEH.170.03.2023). Wistar rats were used with the following criteria: Inclusion criteria: male rats, a weight of 150–250 g, healthy rats, and no defects or wounds on the body. Exclusion criteria: injured during the study, death by pinching, fighting or cannibalism, or other causes.

Experimental animals were grouped into three groups. Each group was given a quercetin pulmospheres labeled with Rhodamine B. A number of animals was determined by number of replications based on Federer’s formula. Animals were acclimatized to room requirements of 22°C ± 3°C, 30%–70% relative humidity, and 12 h of bright 12 h of dark lighting and feeding according to standard and given indefinitely. The weight of the animal should not be more than 20% of the average weight.[20]

Administration of pulmospheres preparation by inhalation

Administration of pulmonary preparations to animals was carried out with nose-only exposure using tools described by Kaur et al., as shown in Figure 1.[21]

Figure 1.

Method of nose-only administration of preparations[21] (a) A tapered poly(propylene) centrifuge tube of capacity 15ml that formed the powder fluidization or aerosol generation chamber; (b) Flexible, C-Flex tubing to introduce a turbulent fluidizing air stream into the chamber; (c) a rubber pipette bulb that provided the source of turbulent air when pressed and released

Administering dose

The daily human dose of quercetin supplement is 250–600 mg.[22] Therefore, the dose of quercetin for experimental animals was converted using the equation:

AED(mg / kg) = Human dose(mg / kg ) × [23]

Notes:

AED = Animal equivalent dose

Km ratio from humans (body weight = 60 kg) to rat experimental animals (body weight = 0.15 kg) is 6.2; therefore AED value is:

Number of preparations given to experimental animals can be calculated using equation:

Procedure for sample administration

Dry pulmosphere samples were weighed and placed on a tube lid, and animals were removed from cages, muzzles directed into tube hole, and left exposed to pulsed powder for 60 s.

Fluorescence imaging

Quercetin pulmospheres with Rhodamine B were used to detect drug deposition in animals using a fluorescence microscope. Animals were sacrificed, lungs were removed, and blood was cleaned using 0.9% NaCl. The organ was homogenized with a formamide-water mixture, incubated for 14 h, and then centrifuged at 4000 rpm for 30 min, allowing for observations.

Quantitative calculation

The study used the McLoughlin method to analyze release rate and in vivo deposition in animals.[24] The animals were divided into three groups and administered different formulas, with organ harvesting occurring at 1 and 6 h postadministration.

Data analysis

The study analyzed the release rate of the drug and its in vivo deposition using one-way ANOVA and the honest significant difference (HSD) Test.

RESULTS AND DISCUSSION

In vitro release of quercetin from pulmospheres

Quercetin release test was conducted for 10 h at physiological lung pH and evaluated release profiles to determine the difference and similarity factors using the DDSolver application [Table 1].[25]

Table 1.

Difference factor (f1) and similarity factor (f2) between formulas

Formula	Difference factor (f1)			Similarity factor (f2)
	Formula 1	Formula 2	Formula 3	Formula 1	Formula 2	Formula 3
Formula 1	0.00	28.67	16.37	100.00	55.49	64.75
Formula 2	22.28	0.00	9.55	55.49	100.00	74.86
Formula 3	14.07	10.56	0.00	64.75	74.86	100.00

The release profile of formulas F1 and F2, F1 and F3 differs due to f1 values above 15, but all formulas have the same release profile (f2 = 50–100).[26]

Formulas F2 and F3 show smaller detached quercetin than formula F1 due to reduced burst effects and improved carrageenan polymer ratio [Figure 2]. Formula F2 has the biggest particle size (2.750 ± 0.141 µm), increasing the polymer layer around drug particles and decreasing the number of released drugs. Formula F2 showed the least amount of quercetin release for 10 h because of the largest particle size and thicker polymer layer around drug particles, resulting in a reduced burst effect and longer drug diffusion out of the pulmosphere.[27,28] In vitro release tests may not accurately represent lung physiological conditions due to the small fluid volume, pH, and mucoadesive properties of polymers. Future recommendations may be based on pharmacokinetics studies.

Figure 2.

Quercetin cumulative release from pulmospheres

Profile and kinetics of quercetin release from pulmospheres

Quercetin content released at each sampling time from in vitro release was included in several drug release kinetics model equations as shown in Figure 3.

Figure 3.

Quercetin release kinetics: (a) Zero order, (b) First order, (c) Higuchi, (d) Korsmeyer–Peppas

The Higuchi model was found suitable for analyzing release kinetics in the quercetin-alginate-carrageenan pulmospheres due to its simple, realistic model demonstrating a direct proportionality between cumulative drug released and the square root of time, and its equation shows a correlation coefficient closest to 1.[29] Drug release from alginate and carrageenan matrix involves swelling, dissolution, and degradation due to damage to calcium alginate’s egg-box structure, and coil-helix structure bond breakdown.[30,31,32]

Release rate from pulmospheres

The study found that an increase in the carrageenan polymer ratio significantly impacted the rate of quercetin release from the pulmosphere. Post hoc Tukey HSD analysis revealed that a 1:2 alginate-carrageenan polymer ratio reduced quercetin release. Three formulas showed sustained release, with a range of 50.47%–58.37% for 10 h. In comparison with Rani et al., they observed the release of isoniazid at 75.11%–94.02% for 10 h from alginate-carrageenan microspheres.[33] Although the sustained release was also achieved, the current results of quercetin showed more sustained and less burst than isoniazid. The release kinetics showed that the isoniazid microspheres followed the Korsmeyer–Peppas model.[33] Therefore, an in vivo release test involving animals is required to ascertain the precise rate of quercetin release from the pulmospheres. Furthermore, the study of a sufficient amount for pulmonary using HPLC analysis is suggested for further research.

Drug deposition

Fluorescence imaging

The study used an inverted fluorescence microscope (Nikon Eclipse Ts2R) to observe rat lung tissue after 1 and 6 h of pulmospheres administration, observing the presence of Rhodamine B and its luminescence intensity at 200× magnification and red filters on the left and right lungs [Figures 4 and 5].

Figure 4.

Drug deposition in the right lung harvesting at 1 h and 6 h after administration

Figure 5.

Drug deposition in the left lung harvesting at 1 h and 6 h after administration

After observation, intensity of Rhodamine B luminescence was observed using Nikon NIS-Elements Advanced Research [Table 2].

Table 2.

Rhodamine B luminescence intensity in the lung

Formula	Average intensity (%)
	Left lung (h)		Right lung (h)
	1	6	1	6
F1	252.667±2.517	251.333±1.528	255.333±3.214	251.667±4.041
F2	168.667±1.528	167.333±2.082	170.667±3.055	166.667±3.786
F3	202.667±6.429	201.333±3.214	204.667±4.163	206.333±5.508

The statistical analysis using two-way ANOVA found no significant difference in rhodamine B intensity between the 1^st and 6^th h of treatment in both the right and left lung. However, pulmospheres showed significant differences. Formulas F1 and F2 showed higher luminosity intensity, while formula F2 had the smallest value [Table 2]. This suggests that increased rhodamine B concentration increases luminescence intensity.[34] The amount of drugs released and rate of release of formula F2 were lowest compared to formula F1 and F3.

Quantitative analysis

The concentration of rhodamine B contained in each lung weight after treatment for 1 h and 6 h can be seen in Table 3.

Table 3.

Rhodamine B concentration in lung

Formula	Rhodamine B concentration (μg/g)
	Left lung (h)		Right lung (h)
	1	6	1	6
F1	18.795±0.74	16.444±3.219	12.490±1.535	11.334±1.379
F2	11.934±2.751	12.364±3.070	8.685±2.672	6.987±1.931
F3	14.522±2.638	14.394±2.620	10.102±2.102	8.640±0.973

Again, the statistical analysis using two-way ANOVA found a decrease in rhodamine B concentration between 1^st and 6^th h in both lungs, but no significant decrease was observed. Formula F2 had the lowest concentration in both lungs. All the pulmospheres were effective against Mycobacterium tuberculosis, with formula F2 showing the most optimal results. MIC of quercetin against Mycobacterium tuberculosis, which is 6.25 µg/g.[35] This Quercetin MIC is considered moderate resistance as similar to moderate category to the MIC for isoniazid (0.25–4 μg/ml), rifampin (0.25–8 μg/ml), and rifabutin (0.25–8 μg/ml) for Mycobacterium tuberculosiss.[36] The polymer ratio 1:2 resulted in the largest particle size, affecting drug entrapment efficiency and loading. The polymer ratio in pulmospheres leads to the formation of a double gel system with alginate and carrageenan, causing particle size to increase.[37] However, continuous carrageenan addition decreases alginate content, resulting in smaller particle sizes.[38] This pulmosphere phenomenon influences deposition efficiency in lung lobes, influenced by the shape, density, surface charge, and electrostatics.

Formulas F2 and F3 showed reduced burst effect when the drug was released from pulmosphere. The reduced burst effect is caused by the addition of carrageenan polymer ratios so pulmosphere matrix is more compact and not porous.[31] In addition, larger particle size causes the thickness of the matrix surrounding the drug to be thicker, causing the drug to take longer to diffuse out through the matrix wall so decreases the release rate.[38]

An increase in rhodamine B concentration causes an increase in the intensity of its luminescence and vice versa.[32] The luminescence intensity of rhodamine B in rat lung tissue varies among formulas, with Formula F2 having the lowest intensity due to its lowest drug release rate. However, drug loading remains low, suggesting a strategy to increase Quercetin concentration (above a current concentration of 0.2%) in formulas for future therapeutic efficacy in human lungs. The correlation between intensity and drug deposition is then recommended for further investigation and validation with additional quantification techniques, such as HPLC analysis of lung tissue.

CONCLUSION

Quercetin pulmospheres with alginate-carrageenan polymer ratios (1:1, 1:2, 1:3) deliver quercetin to lungs, with the lowest rate occurring in 1:2 ratios, delivering it above MIC against Mycobacterium tuberculosis.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil