INTRODUCTION:

Pulmonary diseases have posed a significant threat to global health. With the recent COVID-19 pandemic, respiratory diseases have been severely affected worldwide. However, significant obstacles remain in developing safe and effective treatments for lung disorders^1-3. One of the drug candidates that is extensively studied for lung disorders is curcumin⁴.

Curcumin is derived from the tropical and subtropical plant Curcuma longa (turmeric), which grows in India, Indonesia, China, Taiwan, Thailand, and Vietnam. Turmeric is a well-known spice, food coloring, and medicinal herb. Among its beneficial effects, curcumin showed many properties, including anti-inflammatory, antioxidant, anti-fibrosis, antimicrobial, anti-cancer, antiviral, anti-allergic, and anti-asthmatic effects^4-9. Although curcumin has many health benefits, it does have a few drawbacks, including poor absorption, rapid metabolism, and rapid elimination, which result in its low bioavailability. In addition, curcumin is also rapidly degraded into glucuronide and sulfate metabolites^10-12. Oral administration of curcumin has been shown to result in a low tissue concentration required to affect lung diseases^4,13,14.

Drug delivery to the lung through inhalation therapy has long been recognized as the most effective method of administering drugs that act directly on the respiratory system. Inhalation therapy for pulmonary drug delivery has long been recognized as the most effective drug administration method that acts directly on the respiratory system^1,2,15. The administration of drugs through inhalation therapy offers numerous benefits, including the ability to precisely target the intended organ, resulting in a reduction in systemic side effects. Additionally, this method of drug delivery allows for a decrease in the required dosage, which in turn minimizes the likelihood of adverse reactions and enhances patient adherence to treatment. Furthermore, inhalation therapy offers a prompt onset of action and circumvents the initial phase of hepatic metabolism. Additionally, it provides a substantial surface area that can enhance drug absorption efficacy. The respiratory system exhibits minimal enzymatic activity, thereby circumventing the potential degradation of drug particles before their absorption¹⁶. Thus, drug delivery in the form of an inhalation formulation can be a solution for the delivery of curcumin to the lungs.

Several earlier research investigating curcumin as an inhalation treatment had been conducted. Hemmati AA et al. created a nano curcumin formulation using b-cyclodextrin as a stabilizer, which was mixed at 400rpm overnight, freeze-dried, and delivered to mice by nebulization¹⁷. Zhang B et al. synthesized curcumin by dissolving it in hydroxypropyl-cyclodextrin. The resulting formulation was then delivered to the mice's lungs intratracheally⁵. Al Ayoub et al. produced a nebulized curcumin nanoemulsion using limonene and oleic acid¹⁸. The most significant disadvantage of utilizing cyclodextrin in formula production is that its high molecular weight might increase particle size¹⁹. Compared to nanoemulsion, the nanosuspension technology has various benefits, including being more accessible to manufacture, more physically stable, and using fewer or simpler surfactants, making the formulation process simpler. Furthermore, nanosuspension may increase the solubility of medicines like curcumin^19,20.

In the present study, we aimed to prepare a curcumin inhalation nanosuspension formula, characterize the preparation, and investigate the pulmonary concentration of the nebulized formulation in rats. We hypothesized that the resulting curcumin formulation for inhalation is an effective delivery system and can deliver curcumin directly to the rat lungs.

MATERIALS AND METHODS:

Curcumin nanosuspension formulation:

Curcumin powder was purchased from Phytochemindo Reksa, Indonesia, with 95% curcuminoid content. One hundred milligrams of curcumin were dissolved in 5mL of acetone (E. Merck, Germany), while Poloxamer 188 (BASF Co., USA) was dissolved in 200mL milli-Q water. The curcumin solution was then dropped into the poloxamer solution in increments of 50mL every 10 seconds on a magnetic stirrer at 2000rpm and 25°C. Afterward, the solution was left on a magnetic stirrer for several hours at a higher speed to remove the organic solvent used. The curcumin nanosuspension was then homogenized for 40minutes in a high-speed homogenizer (Ultra-Turrax T-25, IKA, Malaysia) at 20,000rpm. The experiment was done for at least three times.

Acetone residue analysis:

The acetone residue test was done in Saraswanti Indo Genetech Laboratory, Bogor, using Headspace gas chromatography was utilized for the acetone residue test. The method used for the analysis was done according to the residual solvents analysis by the US Pharmacopeia, with a lower limit of quantification for acetone of 0.38mg/L.

Particle size and zeta potential measurement:

The suspension's particle size and distribution index were measured using a Malvern Panalytical. The zeta potential was calculated using the electrophoretic light scattering method on a Malvern Zetasizer Nano ZS.

Morphology of curcumin:

A transmission electron microscope (FE1-Tecnai G2 20S-Twin, FEI Company, China) was used to examine the morphology of curcumin nanosuspension.

Determination of curcumin concentration in nanosuspension:

The HPLC system (Waters Alliance 2695 Separation Module, UK) was used to determine the curcumin content of nanosuspension. A 5mm, 150 x 4.6mm Inertsil^TM ODS-4 C18 HPLC column (GL Sciences Inc, Japan) was used. At a 1mL/min flow rate, the mobile phase was a 50:50 (v/v) mixture of potassium dihydrogen phosphate pH 3.5 and acetonitrile. The internal standard used was irbesartan. Waters 2998 Photodiode Array Detector was used for detection at wavelengths of 424nm for curcumin and 245nm for irbesartan. Degassing was accomplished through filtration through a 0.45mm Whatman membrane filter and 10minutes of sonication. The HPLC system was set to 30°C. A standard calibration curve ranged from 2 to 64mg/mL to determine the nanosuspension concentration.

Stability test:

The stability of curcumin nanosuspension was done by determining particle size, polydispersity index, zeta potential, and curcumin concentration at weeks 1, 2, 4, 8, 12, 16, 20, and 24 after storage at 5⁰C, room temperature, or 40⁰C. The stability was tested according to the technical requirements from the ICH Q1A (R2).

Determination of curcumin concentration in lung and plasma after inhalation in the rats:

The chosen curcumin suspension formulation was then tested for its concentration in the lung and plasma following inhalation in the rats. The in vivo experiment was done after approval from the Ethics Committee Faculty of Medicine Universitas Indonesia (KET-517/UN2.F1/ETIK/PPM.00.02/2021).

Five male Sprague Dawley rats weighing 200 – 250 grams were obtained from the National Agency of Drug and Food Control - Animal Breeding Facility. Two weeks before the experiment, the rats were acclimatized. All rats were kept in a room with a constant temperature and humidity, 12hour light and dark cycles, standard pellet food, and access to water ad libitum. Rats were given curcumin nanosuspension inhalation with a dose of 10mg/kg BW (7-8mL) for 20 minutes via an ultrasonic mesh nebulizer (YM-252, China). The rats were sacrificed one hour after inhalation. The plasma and lung tissue were then collected for curcumin concentration analysis.

Blood samples were centrifuged at 2000g for 10 minutes at 4°C to obtain plasma. The lung tissue was rinsed with saline, dried on a filter paper, and weighed. Tissue homogenization was accomplished by preparing 300mg of lung tissue in 500µl of saline solution using an Ultra-Turrax homogenizer, followed by centrifugation at 12.000rpm for 5 minutes at 4°C, and then the supernatant was transferred to new tubes. Curcumin levels in blood plasma and lung tissue were measured using LC-MS/MS at PT. Farmalab Indoutama in Bandung, Indonesia (Waters) used the previous study's method²¹.

RESULT:

Curcumin nanosuspension:

The formulation of inhaled curcumin nanosuspension was created by experimenting with varying formulas. The organic solvent acetone was utilized to dissolve curcumin and yield curcumin nanosuspension. All organic residues were eliminated after dissolving curcumin. Methods for removing acetone residues from the curcumin nanosuspension used in this investigation include a rotary evaporator and increasing the stirring speed and time (Table 1).

We removed acetone residue in two ways: by using a combination of a magnetic stirrer at 600rpm for 2hours with a rotary evaporator for 2.5hours and by extending the time and increasing the speed on the magnetic stirrer to 5hours and 2000rpm. In the final formulation, we used the second method by extending the time and increasing the speed of the magnetic stirrer. This method is more accessible and straightforward and can achieve smaller particle sizes.

After obtaining the best method to eliminate acetone residues, we developed formulas of curcumin nanosuspension for inhalation using an extended duration on a magnetic stirrer. The five best-studied formulations are shown in Table 2. Of the many formulas, the best results were obtained using magnetic stirring and a high-speed homogenizer to incorporate curcumin with organic solvents, acetone, and the stabilizing agent poloxamer 188 (P-188).

Characterization of curcumin nanosuspension:

The speed and duration used in magnetic stirring significantly affect the particle size. In addition, the concentration of stabilizer used also affects the particle size produced. Table 2 shows that using poloxamer 188 0.15% (v/v) with a magnetic stirring speed of 2000rpm for 5hours can produce nanosuspensions with small particle sizes with good polydispersity index (PD) and zeta potential. Out of the best five formulations, the optimal formulation chosen was NSK-5. NSK 5 shows a small particle size with an optimal polydispersity index and does not contain acetone residue. The curcumin nanosuspension dissolves in water homogeneously and without forming drug crystals (Figure 1).

Figure 1 (A). Curcumin in water, (B) Curcumin nanosuspension for inhalation

The particle size distribution and zeta potential distribution of the final curcumin nanosuspension for inhalation are shown in Figure 2.

Figure 2. Particle size and zeta potential distribution of the final curcumin nanosuspension for inhalation formula

Morphology of curcumin nanosuspension:

Upon examination of curcumin nanosuspension morphology with Transmission Electron Microscopy (TEM), it was found that the form of curcumin in nanosuspension was spherical with a size ranging from 100nm to 200nm (Figure 3).

Figure 3: Morphology of curcumin in nanosuspension. (A) Magnification 9900x (B) Magnification 38000x (C) Magnification 43000x

Entrapment efficiency:

The entrapment efficiency of curcumin in nanosuspension was determined using HPLC with a UV detector. It was found that the average efficiency of curcumin in the nanosuspension was 90.11±6.72% compared to the weighed curcumin.

Stability of curcumin in nanosuspension:

The stability test at 5C showed a slight increase in particle size, but until the 24^th week, the particle size was still below 600nm. The PDI value experienced a slight increase, especially in the 2^nd week, but until the 24^th week, the increase in the PDI value only reached 0.5. The zeta potential also experienced a slight increase and fluctuated (Fig. 4 A, B, and C). The decrease in NSK levels at the end of the first week was 8%; at the end of the 4^th week, the decrease in NSK levels was 7%; at the end of the 8^th week, there was a final decrease of 15%, at the 12^th week there was a decrease of 19% and at the end of week 24 decreased by 22% (Fig. 4D).

The stability test at room temperature showed a slightⁱncrease in particle size, but until the 24^th week, the particle size was still below 600nm. The PDI value has increased from the 2^nd week until the 24^th week; the increase in PDI is still below 0.5. The zeta potential value has increased and fluctuated (Fig. 4 A, B, and C). The decrease in NSK levels at the end of the first week was 5%; at the end of the 4^th week, the NSK levels decreased by 12%; at the end of the 8^th week, there was a 15% decrease, at the end of the 12^th week it decreased by 17% and at the end of week 24 decreased by 21% (Fig. 4D).

The accelerated ^stability test at 40C showed a higher increase in part^icle size compared to other temperatures, but until the 24^th week, the particle size was still below 800 nm. The increase in PDI exceeded 0.5 but was still below 0.7. The zeta potential value also increased and fluctuated (Fig. 4A, B, and C). The decrease in NSK levels at the end of the first week was 18%; at the end of the 4^th week, the NSK levels decreased by 15%; at the end of the 8^th week, there was a decrease of 17%, at the end of the 12^th week it decreased by 18% and at the end of week 24 decreased by 25%. (Fig. 4D).

Figure 4. Stability of curcumin nanosuspension after storage up to 24 weeks

Curcumin concentration in lung and plasma after inhalation in the rats:

The rats were subjected to a nebulization procedure to administer curcumin nanosuspension for inhalation, as depicted in Figure 5. Curcumin was detected in the lung at a concentration of 0.161±0.022ng/100mg of lung tissues (N=5); however, it was not detected in the plasma of the rats.

Figure 5: Curcumin nanosuspension was administered to the rats via a nebulization procedure.

DISCUSSION:

Pulmonary drug delivery by spraying medications directly into the trachea is an appealing way of administration. Due to its direct delivery method, inhalation therapy is the most effective treatment for lung illnesses. Curcumin is one of the drug candidates that is being investigated intensively for lung disorders.⁴ In the present study, we successfully developed a formulation of curcumin nanosuspension using a high-speed homogenization (HPH) technique, with proven local delivery to the lung and undetected in the systemic circulation. A nanosuspension is a type of colloidal dispersion that contains drug particles that are submicron in size, measuring less than 1 µm. Nanosuspension production involves specific techniques, and stabilization is achieved through appropriate stabilizing agents. Numerous methods exist for nano-suspension production, including media milling, antisolvent precipitation, precipitation ultrasonication, flash nanoprecipitation, high shear homogenization, high-pressure homogenization, and mixed techniques²². Among the available techniques, high-pressure homogenization is the most common method to prepare nanosuspension. However, high-pressure homogenization needs a longer step, such as pre-processing and micronization of the drug. Further, the use of expensive equipment is essential for the method, resulting in a higher cost of the dosage form²³. Thus, in the present study, we used high-speed homogenization instead of high-pressure homogenization because this tool is more accessible, straightforward, and inexpensive.

The first step of developing curcumin nanosuspension for inhalation is ensuring solubility. Curcumin exhibits significant solubility in various organic solvents, including dimethoxy sulfoxide (DMSO), ethanol, methanol, chloroform, and acetone²⁴. Due to its facile evaporation and suspension removal properties, acetone was selected as the solvent for curcumin dissolution²⁵. Based on available data indicating that exposure to acetone concentrations below 1,000 parts per million only results in mild irritation, it is advisable to establish a threshold limit value-time weighted average (TLV-TWA) concentration of 750ppm for acetone. Studies conducted on humans to assess the respiratory impact of exposure to inhaled acetone have predominantly reported irritation in the nasal, pharyngeal, tracheal, and pulmonary regions²⁵.

The other crucial component to producing a good quality nanosuspension is stabilizers. Improper utilization of the stabilizer may result in the aggregation of drug crystals due to the high surface energy of the nanoparticles. The primary role of the stabilizer is to uniformly coat the drug surface, thereby inhibiting Oswald's ripening and preventing the aggregation of nanosuspension particles. Using a stabilizer results in a physically stable formulation, which is achieved by creating a steric or ionic barrier²⁶.

In our study, poloxamer 188 was used to develop curcumin nanosuspension due to its excellent stabilizing properties. Poloxamer 188 is a nonionic copolymer comprised of a hydrophobic polypropylene oxide (PPO) chain and two hydrophilic polyethylene oxide (PEO) blocks. The aqueous solubility of the substance is due to its PEO block, which facilitates the formation of hydrogen bonds between the oxygen moieties of the substance and the water molecules. The PPO typically generates a centralized hydrophobic core, where the methyl groups interact with dissolved substances through van der Waals forces. Poloxamer 188 exhibits the property of solubility in both polar and nonpolar solvents²⁷. In addition, poloxamer 188 had been shown to have a low risk of toxicity for inhalation administration. It did not cause any changes or immune responses in pulmonary histology²⁸. A study by Lindberg et al. in an in vitro study using BE-AS-2B cells, human immortalized bronchial epithelial cells, demonstrated that poloxamer 188 was nontoxic at high concentrations compared to polysorbate 20 and polysorbate 80²⁹.

Upon the formation of the nanosuspension, it is essential to characterize the formulation, specifically regarding the average particle size and its distribution, polydispersity index, and the zeta potential, representing the particles' electric charge, to ascertain the nanoparticles' physical stability. The particle size of nanosuspension is significantly influenced by the speed and duration of time utilized during magnetic stirring. Additionally, the particle size generated is influenced by the concentration of stabilizer applied²². The incorporation of poloxamer, coupled with an increased stirring rate over 5 hours, yielded an optimum nanosuspension characterized by small particle sizes, satisfactory polydispersity index, and zeta potential. In this study, our curcumin nanosuspension resulted in a particle size of 281.3nm with a PDI of 0.464 and a zeta potential of -29.4mV. The size of the nanosuspension particles is affected by the speed and duration used when in the magnetic stirrer. In addition, the particle size formed is affected by the concentration of the stabilizer used²². To obtain an optimal curcumin nanosuspension, we use poloxamer-188 0.15% (v/v) with magnetic stirring at 2000rpm for 5hours, followed by HSH 20.000rpm for 40 minutes. Determination of the particle size in nanosuspensions is essential in assessing the drug's ability to reach the desired target effectively. The parameter used is the Z-average, indicating the particle's hydrodynamic size parameter. Z-average is the most important and stable variable in examining dynamic light scattering. The Z-average is an intensity-weighted distribution in which the contribution of each particle to the distribution is related to the light scattered by the particles³⁰. The polydispersity index (PDI) is a parameter that characterizes the size of the particles in the sample. The PDI value is between 0 and 1, where 1 represents a very heterogeneous population, and 0 represents a very homogeneous population³¹. A good PDI ranges from 0.05 to 0.7. A PDI value of more than 0.7 indicates a wide distribution of particles³².

The zeta potential serves as an indicator of the enduring stability of colloidal suspensions. A more significant zeta potential corresponds to heightened resistance to aggregation and increased suspension stability. A zeta potential of at least ±30mV. However, a minimum zeta potential of ±20mV is essential to achieve optimal stability when electrostatic and steric stabilization methods are employed³³. The nonionic stabilizer utilized in this study, poloxamer 188, can furnish steric stabilization in suspension, thereby ensuring the stability of the nanosuspension²⁹.

The crystal structure and particle morphology analysis are essential in understanding the potential polymorphic or morphological alterations in the drug upon reaching the nanoscale level²². Our formulation's transmission electron microscopy (TEM) analysis revealed that the resulting curcumin particles exhibited a spherical morphology and uniform distribution. The drug and stabilizer solution's temperature and mixing rate affect the particle morphology. The temperature must be kept low. At high temperatures, there will be rapid removal of organic solvents, which will produce irregular particles. If the temperature is kept low during formulation, the solvent will leave the solution at a low rate, forming spherical and uniform nanoparticles. The mixing rate of the drug solution and the solution containing the stabilizer must be kept constant to form uniform and spherical particles³⁴. Concerning the dispersibility of a suspension containing nanoparticles, spherical particles exhibited superior disintegration characteristics and greater nanoparticle liberation when utilized in a pulmonary sprayer, as opposed to cylindrical particles³⁵.

The curcumin nanosuspension exhibits satisfactory entrapment efficiency and demonstrates good stability for three months when stored at room temperature. Furthermore, our formulation has displayed localized distribution specifically to the lung without entering the systemic circulation.

In the stability test of curcumin nanosuspension at the end of the second week, there was a slight increase in particle size, PDI, and zeta potential at 5C and room temperature, with the most significant increase at 40C. However, until the 24^th week, the particle size was still in the nano range. The increase in particle size and zeta potential was due to the decrease in the steric force caused by poloxamer 188, causing particle agglomeration. Increasing the storage temperature causes a faster size increase. This is because an increase in temperature causes particles to move faster, leading to increased agglomeration³⁶. The increase in PDI shows that NSK is increasingly heterogeneous. However, the PDI increase at all temperatures was still below 0.7, indicating that the polydispersity of NSK was still good.

Curcumin levels decreased at the end of the first week, especially at 40 ^oC storage. At the end of the 24^th week, curcumin levels decreased by 21-25% at the three temperatures, with the most significant decrease at 40 ^oC. Mirzaee F et al. stated that curcumin degradation increased with increasing temperature³⁷. There is limited data on curcumin nanosuspension formulations that inhalation can administer. A previous study by Hemmati et al. also successfully produced a freeze-dried particle for nanosuspension formulation. Thus, their curcumin requires dissolution before nebulization¹⁷. In the present study, our formulation does not need a dissolution procedure before use, so it is easier to apply. In addition, the present production method is easy, fast, and cost-effective.

Finally, the present formulation of curcumin nanosuspension exhibits satisfactory entrapment efficiency and demonstrates good stability for three months when stored at room temperature. Furthermore, our formulation has displayed localized distribution specifically to the lung without entering the systemic circulation. Nonetheless, administering drugs through inhalation necessitates appropriate calibration to achieve the intended therapeutic objective, whether to produce systemic outcomes or solely localized results.

CONCLUSION:

A curcumin nanosuspension formulation for inhalation was developed through poloxamer 188 and subsequent high-speed homogenization. The preparation process was fast and uncomplicated. Furthermore, the formulation had shown efficacious when given via nebulization for targeted administration to the lung. Compared with previous studies, this curcumin nanosuspension formulation is proven that it can be administered locally to the lungs with minimal systemic effects.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this present study.

ACKNOWLEDGMENTS:

This study was supported by a Grant from the Indonesian Ministry of Education and Culture No NKB-919/UN2.RST/HKP/05.00/2022 and NKB-880/UN2.RST/HKP/05.00/2023.

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Received on 10.04.2024 Revised on 12.07.2024

Accepted on 10.10.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1757-1764.

DOI: 10.52711/0974-360X.2025.00252

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Formulation Code	Speed on magnetic stirrer (rpm)	Duration of magnetic stirring	Speed and duration of homogenization	Duration on the rotary evaporator	Acetone residue (mg/L)
NSK-A1	600	2 hours	15000 rpm 20 min	-	17 449.8
NSK-B1	600	2 hours	20000 rpm 40 min	1 hour	443.4
NSK-C1	600	2 hours	20000 rpm 60 min	2.5 hours	Not detected
NSK-A	600	4 hours	20000 rpm 40 min	-	1065.5
NSK-B	1250	4 hours	20000 rpm 40 min	-	415.7
NSK-C	2000	5 hours	20000 rpm 40 min	-	Not detected

Formulation Code	Stabilizer	Speed (rpm)	Duration of magnetic stirring	Zeta Potential (mV)	Poly-dispersity index (PDI)	Particle Size (nm)
NSK-1	Poloxamer 188, 0.15%	700	4 hours	-20.3	0.257	243.4
NSK-2	Poloxamer 188, 0.1%	800	4 hours	-22.4	0.308	210.9
NSK-3	Poloxamer 188, 0.1%	1250	4 hours	-31.3	0.439	414
NSK-4	Poloxamer 188, 0.1%	2000	5 hours	-21.2	0.394	586.3
NSK-5	Poloxamer 188, 0.15%	2000	5 hours	-29.4	0.464	281.3