ABSTRACT

Objectives

Bortezomib (BTZ) functions as an androgen receptor signalling inhibitor, is used for the treatment of prostate cancer, and has been sanctioned by the United States Food and Drug Administration. The medicinal applications of BTZ are impeded by low solubility, first-pass metabolism, and restricted bioavailability. This study aimed to develop and enhance polylactic acid-co-glycolic acid (PLGA) nanobubbles (NBs) as a sustained-release mechanism for BTZ, thereby augmenting stability and bioavailability.

Materials and Methods

Seventeen experimental runs were conducted to optimize drug-PLGA NBs using a three-factor, three-level Box-Behnken Design. The improved formulation comprised 30 mg of medication, 250 mg of PLGA, and 2.0% w/v polyvinyl alcohol as a stabilizing agent.

Results

The NBs exhibited a particle size of 186.9±13.9 nm, a polydispersity index of 0.146±0.042, and a zeta potential of -21.4±2.28 mV, along with an entrapment efficiency of 66.12±1.48%. Fourier transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction analysis verified the absence of drug-polymer interactions, whereas scanning electron microscopy demonstrated uniform spherical nanoparticles. In vitro experiments demonstrated superior drug release, and stability assessments indicated no major alterations after one month. Pharmacokinetic studies in rats demonstrated an elevated C_max (1.69) and area under the curve from time 0 to t (1.63), signifying enhanced sustained release and absorption. The results underscore the capability of BTZ-loaded PLGA NBs to augment drug kinetics and bioavailability, hence facilitating targeted distribution and enhanced therapeutic efficacy.

Conclusion

This investigation offered significant insights into the factors influencing oral absorption in NB formulations, which can guide future methods for oral medication development. BTZ-loaded PLGA nanobubbles showed promising results by enhancing oral absorption and improving pharmacokinetics in the study, which points to their potential use in sustained-release drug delivery. These findings offer a stepping stone toward nanomedicine via the oral route in future drug development.

INTRODUCTION

B lymphocytes are a type of white blood cell that is impacted by multiple myeloma (MM), a cancerous condition. It is identified by the aberrant proliferation of a single bone marrow-derived plasma cell that produces monoclonal immunoglobulins.¹ MM is the second most common haematological malignancy, accounting for 1% of all cancers and 13% of haematological cancers.^{2, 3} One promising approach for treating MM is targeting the ubiquitin proteasome system (UPS).⁴ The UPS is responsible for degrading defective proteins by recognizing poly-ubiquitin chains attached to them. Inhibiting proteasome activity leads to the accumulation of cells, triggering apoptosis or programmed cell death. Proteasome inhibitors have shown potential as antitumor agents and can also inhibit angiogenesis (anti-angiogenic properties) by blocking the UPS. Bortezomib (BTZ) (Velcade^®), an approved drug by the Food and Drug Administration, is commonly used as a first-line treatment for MM and mantle cell lymphoma.⁵ By acting as a proteasome inhibitor, BTZ binds reversibly to the chymotrypsin-like subunit of the 26S proteasome. This mechanism inhibits the proteasome, ultimately halting the breakdown of various pro-apoptotic factors.⁶ BTZ is offered in intravenous (IV) and subcutaneous formulations. Mannitol, at a concentration of 10 mg per gram of BTZ, is used to improve solubility upon reconstitution (Velcade^®). The sugar molecule helps in BTZ dissolution by forming a BTZ trimer and boronate ester, through covalent binding with the boronic acid moiety of BTZ. This ester prevents trimer formation, which could reduce water solubility. Moreover, mannitol, a highly water-soluble polyol, lowers the pKa of BTZ by three units, further increasing water solubility.⁷

The main issue with the current formulation of BTZ, which utilizes mannitol for solubility enhancement, lies in its lack of suitability for long-term drug delivery. Mannitol aids in BTZ dissolution and increases water solubility. However, its use as a cosolvent in the formulation may pose challenges for sustained drug release over an extended period. Mannitol’s rapid dissolution and clearance from the body may lead to a shorter duration of therapeutic effect, necessitating frequent administration to maintain therapeutic levels of BTZ. Therefore, alternative formulations or delivery strategies that ensure sustained release and prolonged therapeutic efficacy may be required for long-term use of BTZ.

Despite the favourable clinical results, the use of BTZ has been limited because it depends heavily on proteasome function, leading to toxicity. Moreover, the medication is linked to various adverse reactions.^{5, 8} Additionally, BTZ is faced with pharmaceutical challenges due to its low water solubility (pKa value of 13) and a brief half-life in the bloodstream.⁵ The combination of toxicity and unfavourable pharmaceutical characteristics creates substantial barriers to its use in clinical settings. As a result, there is an urgent need for a specialized formulation of BTZ. The various physicochemical and pharmacokinetic properties of BTZ are listed in Supplementary Table 1.

Therefore, a strategy that reduces the pharmacokinetic variations between the fed and fasting states must be developed. Strategies such as liposomal formulations, polymeric particles, and self-microemulsifying drug delivery systems are designed to improve the bioavailability of the drug.^9-11 In vivo performance, scalability issues, and the need for costly and specialised branded excipients are the obstacles with the reported approaches. Beyond enhancing solubility, an approach that offers the potential to target drug molecules specifically to diseased tissues while reducing their concentration in normal tissues is essential. Such a targeted delivery system will improve stability in bodily fluids, optimize concentration and release kinetics in the bloodstream, and enhance pharmacokinetic and pharmacodynamic properties. All these factors collectively contribute to heightened efficacy and reduced side effects. One example is the Smart delivery system, which is currently focused on cancer treatment.

At the forefront of these innovative delivery systems are smart delivery systems, which have garnered significant attention and focus, particularly in the realm of cancer treatment. By leveraging advanced technologies and principles, delivery systems that are precisely targeted and regulated represent a state-of-the-art method for administering medication aimed at navigating drugs to their intended targets within the body. Through the integration of sophisticated mechanisms, these systems hold the potential to revolutionize cancer therapy by maximizing efficacy and minimizing the impact on healthy tissues.

A smart drug delivery system possesses the ability to react to sudden changes in its surroundings, particularly those brought on by chemical stimuli. Extensive research has been conducted on the application of pressure waves and ultrasonic (US) triggers in drug delivery systems, which are dependent on external stimuli.¹²

The potential for targeted therapy in a range of medical applications has been enhanced by the broad exploration of these external stimuli to establish precise control over medication release.

Nanobubbles (NBs), or microscopic bubbles at the nanoscale, are used in numerous fields, most notably in drug delivery systems. Their amazing stability, high internal pressure, and large surface-to-volume ratio are only a few of their outstanding physical characteristics.¹³

Drugs and other therapeutic agents can be efficiently delivered to cancer cells by using NBs as carriers. NBs can vary in size from 1 nm to 500 nm, and their dimensions can be precisely tailored to the nanoscale using amphiphilic polymers such as Pluronic or surfactants. By improving blood-brain barrier penetration, NBs provide opportunities for focused drug delivery in neurological diseases, including Alzheimer’s and Parkinson’s. They also show potential in cardiovascular illnesses for precise thrombolytic therapy, enhancing clot breakup with fewer side effects. Research has been done on both polymeric and lipidic NBs; however, the instability of lipid-based NBs leads to breakdown and a shorter circulation period. Poly (lactic-co-glycolic acid), or PLGA, is a special kind of nano/micro biomaterial that can be used for diverse applications such as targeted medication delivery, molecular diagnostics, tissue engineering, and gene transfer.¹⁴ Its high stability, biodegradability, and ease of chemical modification are the favourable characteristics of the PLGA.¹⁵

To the best of our knowledge and based on available literature, there are no prior studies documenting the utilization of PLGA NBs for delivering BTZ to cancer cells. This study aimed to develop and enhance polylactic acid-co-glycolic acid (PLGA) NBs as a sustained-release mechanism for BTZ, thereby augmenting stability and bioavailability. The research involves the preparation of BTZ-containing NBs using PLGA, followed by comprehensive in vitro characterization, including analysis of particle size (PS), size distribution, zeta potential (ZP), morphology, release kinetics, and subsequent in vivo evaluation.

MATERIALS AND METHODS

The pure drug BTZ was acquired from Dr. Reddy’s Ltd., Hyderabad, India. Sigma Aldrich, USA, supplied Poly (D, L-lactide-co-glycolide) 50:50 with an intrinsic viscosity of 0.22 dL/g and a molecular weight of 25,000. Polyvinyl alcohol (PVA; Mw 30,000-70,000) was purchased from Sigma Aldrich (St. Louis, MO, USA). Borviz 2 injection was purchased from Intas Pharmaceuticals Ltd., Ahmedabad, India. Antipyrine was procured as a sample from M/s Yarrow Chemicals Pvt., Ltd., Hyderabad. Isopropanol and dichloromethane (DCM) were acquired from S.D. Fine Chemicals, Hyderabad. We purchased acetonitrile from Qualigens, India. The cells were purchased from NCS Pune, Maharashtra, India. All the media components were purchased from Gibco, USA, and Invitrogen, USA.

Analytical method development using reverse-phase high-performance liquid chromatography

Chromatographic analysis of BTZ was executed using a Shimadzu Prominence model LC-20 AD, equipped with an ultraviolet detector set at 230 nm. A reverse phase Luna C-18 column (150 mm × 4.6 mm i.d., 5.0 μm PS and 100 Å pore size) maintained at 40.0±0.1 °C was employed. The mobile phase is composed of a mixture of acetonitrile and ammonium phosphate (buffer pH 4 adjusted with acetic acid) (60:40 v/v). The mixture was sonicated for 45 minutes for degassing purposes and then filtered through a 0.45 μm pore diameter Whatman filter paper. The flow rate was adjusted to 1.0 mL/min. A standard stock solution of the drug at a concentration of 1000 μg/mL was prepared, followed by the creation of various working standard solutions ranging from 0.5 to 100 μg/mL through serial dilutions. A stock solution (1000 µg/mL) of Antipyrine as IS (internal standard), after appropriate dilution, was included in the experiment. Retention time of drug and internal standard was 4 minutes and 6.8 minutes, respectively.¹⁰

BTZ-NBs formulation development and optimization

BTZ-loaded PLGA NBs were synthesized using solvent evaporation with ultrasound assistance following a reported method with slight modifications. Initially, a homogeneous solution was formed by dissolving PLGA (200 mg) in a water-immiscible solvent, DCM. The drug BTZ was then added to create a dispersion, which underwent sonication for five minutes at 45% amplitude, in an ice bath using a Digital Sonifier S-250D (Branson US, Danbury, USA). Next, the drug dispersion was combined with 25 mL of chilled 2.0% PVA solution and homogenized at 13222 rpm for 10 minutes using a high-speed homogenizer. The formulation was then subjected to sonication using a US probe at 30 W for 3 minutes. A 2.5% v/v isopropanol solution (25 mL) was added to the emulsion and mechanically stirred for 5 hours to remove the DCM. Subsequently, the resultant product was centrifuged at 8000 rpm for five minutes. The product was washed with distilled water after discarding the supernatant. This centrifugation and washing process was repeated three times. The NBs were freeze-dried for 36 h without exposure to light using a LYPH LOCK 4.5 (Labconco Corporation, Kansas City). Later, C3F8 gas (perfluoropropane) was added to the lyophilization chamber through a vial connector at a flow level of 50 mL/min for 1 minute. After this, the screw vials were tightly sealed for further analysis.¹⁶ BTZ-loaded PLGA NB optimization was achieved by implementing a three-factor, three-level BBD. A total of 17 experimental runs were conducted, including three replicated centre points.¹⁷

The three independent variables were the amount of PVA (w/v), homogenization speed (rpm), and homogenization time, each at three levels of variation: low (-1), middle (0), and high (1). The response (three dependent variables) were PS (Y1), polydispersity index (PdI) (Y2), and encapsulation efficiency (% EE) (Y3); Table 1 lists their respective ranges. Utilizing response surface charts and contour (2D) plots, the response surface search was carried out with Design Expert^® (Version 12.0.3.0, Stat-Ease Inc., Minneapolis, MN).

Characterization and evaluation

Measurements of PS, PdI, and ZP

A Zetasizer (Malvern Instruments, UK) was used with DLS theory (dynamic light scattering) to calculate the PS, PdI, and ZP of BTZ NBs after tenfold dilution of the sample with double-distilled water.¹⁸

Percentage entrapment efficiency (% EE)

The % EE of the drug in the NBs can influence the therapeutic efficacy, stability, and release kinetics of the loaded compounds within the NBs. DCM was used to dissolve a particular quantity of NBs containing the loaded drug (BTZ) NBs. The complex was dissolved by subjecting the solution to sonication for 10 minutes. The resultant solution was then suitably diluted and assessed using the above-mentioned HPLC method.

The following equation was used to calculate the same:

Morphology using scanning electron microscopy (SEM)

Using a Quanta FESEM 250, the structure of the NBs and the drug was captured. Before testing, the sample was mounted on an aluminium pin stub after being mounted with double-sided carbon tape and Au-sputter coated utilizing an ion sputter. The specimen was then analysed at an operating distance of 10 millimetres, with an acceleration current of 30 kV and a magnification of 500-10,000 times.¹⁹

Fourier-transform infrared (FTIR) spectroscopy

The spectrum of FTIR was obtained using a Perkin-Elmer spectrometer (Model 1600; USA). The pure drug, PLGA, physical mixture (PM), and the optimized drug-loaded NBs were all analysed at wave numbers 4000-450 cm-1 with a resolution of 1.0 cm-1.²⁰

Differential scanning calorimetric study and X-ray diffraction pattern (XRD)

DSC (DSC-60, Shimadzu Corp., Japan) was employed to ascertain the drug’s physical structure and the potential for chemical interactions with the excipients. Samples of 3-5 mg (pure drug, blank NBs, PLGA, optimized drug-loaded NBs, and NBs stored for three months) were subjected to heating (50-400 °C, 5 °C/min) in folded aluminum pans in a nitrogen environment before being subjected to DSC analysis, following calibration using Indium and Lead standards. The melting point (MP) and the enthalpy of fusion were computed automatically. The X-ray diffraction patterns of the pure drug, the PM, and the optimized formulations were obtained using a Philips X-ray diffractometer (PW-1710) operated with a graphite monochromator and Ni-filtered Cu Ka radiation (at 100 mV and 40 kV). The samples were scanned between 2° and 60° 2 theta (q) angle with an average step size of 0.045° and a duration per step of 0.5 seconds.²¹

Drug release

The release studies of pure drug and optimized drug-loaded PLGA NBs were carried out using in vitro experiments with a shake flask equipped with a dialysis bag. Following encapsulation in dialysis membranes, the specimens were transferred into a conical flask containing phosphate buffer (pH 7.4), maintained at 37 °C, and subjected to constant rotation at 100 rpm. One mL of sample was removed from the outer solution and replaced with brand-new PBS at pH 7.4 at predefined intervals. These aliquots were filtered and analysed using the HPLC method at 230 nm to measure drug release. Three duplicates of the experiment were carried out.²²

Stability studies

The optimized formulation’s stability was evaluated by storing it at three temperatures (4 °C, 25 °C, and 40 °C) at 75% relative humidity. Regular intervals were set to measure PS, PdI, and EE% % changes in the samples.²³

Pharmacokinetic studies (PKs)

Male Wistar rats with an approximate weight of 200±20 g and an age of 4-5 weeks were procured from the National Institute of Nutrition situated in Telangana, India. The animal protocol was designed and approved by the Institutional Animal Ethics Committee of Teegala Ram Reddy College of Pharmacy (approval number: 1447/PO/Re/S/11/CPCSEA-97/A, dated: 17.02.2024). For about a week, animals were exposed to natural light/dark settings, acclimating to a relative humidity of 40–60% and a temperature of 20 °C±2 °C. Then they were split into three groups of six animals randomly. An optimized drug-loaded NB (0.2 mg/kg BW) and the pure drug (dispersed in 0.5% w/v carboxymethyl cellulose) were administered by the oral route, while the reference (marketed product) was given by the IV route. The animal blood was obtained from the retroorbital plexus (300 µL) and then transferred into sterile test tubes with EDTA at specific intervals (0.25, 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 h). Blood samples were centrifuged at 7500 rpm for ten minutes using an Eppendorf centrifuge. The resulting plasma was further analysed using HPLC.¹⁰

Using the protein precipitation method, the drug was recovered from plasma samples. By adding 250 µL of acetonitrile to 50 µL of rat plasma and vortexing the mixture, the drug was effectively extracted from the plasma. After centrifuging the supernatant for 12 minutes at 8000 rpm, chromatography was used to analyze it at a wavelength of 230 nm. Non-compartmental analysis using WinNonlin (version 3.1; Pharsight Corporation, USA) was employed to calculate the C_max (maximum plasma concentration), AUC₀-72 (area under the plasma concentration vs. time curve from 0 to 72 h), T_max (time to reach the maximum plasma concentration), Kel (elimination rate constant), and t1/2 (half-life).

Statistical analysis

Non-compartmental analysis WinNonlin (version 3.1; Pharsight Corporation, USA) was employed to calculate all pharmacokinetic parameters. All the data were expressed as mean ± standard deviation.

RESULTS

BTZ-NBs formulation development and optimization

The study aimed to formulate BTZ NBs by integrating ultrasound technology with solvent evaporation. The drug had been mixed with PLGA that had been dissolved in DCM, sonicated, and added to a cooled 2.0% w/v PVA solution. After that, a 3-minute sonication at 30 W and high-speed homogenization were performed on the sample. Isopropanol solution (2.5% v/v) was used to extract DCM.^{24, 25}

BTZ PLGA NBs formulation optimization by Quality by Design emphasizing preciselyby monitoring critical quality attributes (CQAs) to achieve and maintain QTPP was carried out.²⁶ The current study selected PS, PdI, and EE as CQAs (Table 2). Multiple linear regression analysis (2FI) constructed polynomial models (quadratic, two-factor, and linear). The model selection used R², predicted R², adjusted R², and coefficient of variance (C.V.). Analysis of variance (ANOVA) assessed the impact of variables on responses.

PS

A small size of NBs resulted in a significantly higher surface area-to-volume ratio, enhancing their stability, penetrability, and reactivity in targeted drug delivery 17. After 17 trials, PS ranged from 182.78 to 302.88 nm. The model’s f value of 21.43, with a 0.03 percent chance of being noise, confirmed its “quadratic” nature and that the lack of fit is insignificant. The various perturbation plots of PS, PdI, and % EE are shown in Supplementary Figure 1. ANOVA found variables with p-values below 0.0500 that significantly impacted the response. The “lack of fit f value” (0.77) implied that any lack of fit was not statistically significant. There was a 56.84% chance that a “Lack of Fit F-value” of this extent would occur due to random noise, undermining the model’s reliability. The contour plots (CP) and 3D response surface plots (RSP), illustrating variable influences on PS, were depicted in Figure 1. The R2, corrected R2, and anticipated R2, with values of 0.9650, 0.9200, and 0.7604, respectively, showed a model precision of 13.19, surpassing the required value of 4.

Stabilizer concentration (A), with a high negative coefficient, was the most influential factor affecting PS, as it demonstrated that alterations in this factor resulted in a corresponding change in PS. Stabilizer concentration, homogenization speed, and time, denoted by variables A, B, and C, also played a role in PS, although to a lesser extent than the other factors.

The interaction terms (BC) suggested a combined effect of these variables, and this had a significant impact on PS. the positive coefficients for quadratic terms like A2 and C2 indicate that the squared values of A and C have a nonlinear relationship with PS, potentially leading to changes in PS that are not directly proportional to changes in the variables. Model terms (A, B, C, BC, A², and C²) had p values <0.0500, signifying their effect. The resulting regression equation was:

PS = 217.63 - 28.79 A - 23.80 B - 24.46C+1.01 AB - 0.3550 AC + 19.27B C + 25.67 A²) - 1.98 AB² + 10.10 C²

PdI

The PdI is a dimensionless metric that quantifies the broadness of the PS distribution.¹⁷

Typically, it falls between 0 and 1. Formulations exhibited PdIs from 0.132 to 0.488, with a model f value of 19.68, which indicated a significance of the proposed “quadratic” model and an insignificant lack of fit (f value 0.78). There was a 56.38% chance that a “Lack of Fit f value” of this magnitude would arise due to random noise, underscoring the model’s reliability. Regression coefficients (R2, adjusted R2, and anticipated R2) were 0.9620, 0.9131, and 0.7383, which showed the model’s usefulness with precision exceeding the necessary value (15.5323). Model terms (A, C, A2, and C2) had p values <0.050, signifying a substantial impact. The resulting regression equation is:

PDI = 0.2348 - 0.0876 A + 0.0194 B -0.0430 C-0.0037 AB + 0.0131 AC-0.0105 BC + 0.0440 A² 0.0203 B² + 0.0226 C²

Positive coefficients signify a rise in the associated variable(s), which increases PdI, while negative coefficients indicate a decline, which reduces PdI.²⁷ An increase in variables A and C led to a decrease in PdI, whereas higher values of B (homogenization speed) led to higher PdI. All quadratic terms have positive coefficients, indicating that increases in the squared values of variables A, B, and C lead to increases in the PdI. The response surface and contour plots illustrating variable effects on the PdI are shown in Figure 2.

Percentage entrapment efficiency (% EE)

Impact on % EE ranged from 52.26% to 78.16%. The NB with high entrapment is always desirable, as it helps reduce the dose of the drug.²⁷ The model’s f value, 63.30, with a 0.01% chance, is likely due to noise, indicating significant and negligible fit error for the suggested “quadratic” model. The F-value for lack of fit (1.41) was statistically insignificant based on pure error, with a 36.19% probability of being noise. ANOVA identified significant factors (p value <0.1000), which led to the removal of non-significant variables. The consequence of variables on ZP, as demonstrated by CP and RSP, is shown in Figure 3. The regression coefficients, R2, adjusted R2, and anticipated R2 were 0.9879, 0.9723, and 0.8908, respectively. The predicted R2 aligns closely with the adequate R2, differing by 0.2. The model, evidenced by an adequate precision (signal-to-noise ratio) of 31.5008, surpassing the necessary value of 4, proved helpful in exploring the design space. Figure 4 illustrates a CP and 3D SP, which showcase the influence of selected variables on % EE. An elevation in factors A and B, both related to stabilizer concentration, typically results in decreased entrapment efficiency. In contrast, the interaction terms and quadratic terms increased the percentage of EE. By comprehending the impact of these factors on entrapment, researchers can fine-tune formulations to attain desired levels of efficiency, thereby guaranteeing the effective delivery of substances in pharmaceutical or other applications. Model terms (A, B, AB, AC, BC, A², B², and C²) had p values <0.0500, signifying a significant effect. The resulting regression equation is:

EE% = + 64.01 - 2.51 A - 7.54 B + 0.7489 C + 4.67 AB + 1.63 AC + 2.84 BC + 2.47 A² - 2.37 B² + 0.7209 C²

Characterization and evaluation of NBs

PS, PdI, ZP, % EE, and morphology

PSs and uniformity in the formulation remained consistent, with PS of 186.9±13.9 nm and PdI of 0.146±0.042. A polydispersity value below 0.3 indicated homogeneity. The optimized formulation’s ZP, indicative of colloidal particle surface properties, was -21.4±2.28 mV, with a % EE of 66.12±1.48. The PS and ZP of the optimized NBs are shown in Figure 5. NB stability heavily relies on elevated ZPs, which play a crucial role in maintaining electrostatic repulsion among particles, thus preventing their aggregation. This stability is of utmost importance as it ensures the integrity of NBs during storage and administration, ultimately enhancing their performance in drug delivery and medical imaging applications. PS and ZP were shown in Supplementary Figure 2. It was evident from the figure that the drug had a variety of PSs and a non-uniform cubic shape containing particles as small as micrometres. However, after the formation of the NB, the micronized drug particles consistently changed to spherical particles in the nanosize range.

FTIR

Figure 5 illustrates the determination of component compatibility observed for nano formulation, excipients, and plain drug infrared spectra, using a scanning range of 450 cm-1 to 4000 cm-1. Distinctive peaks in the plain medication were observed at 3446.9; 3294; 2937.68; 1747.57; 1663; 1523; 1460; 1394; 1261.49; 1193.98; 1085.96; 1022; 885.36; and 719 cm-1. There was a physical interaction between the drug and stabilizer, as evident in the stability of the apparent peaks and the absence of new peaks in both the PM and the freeze-dried formulation. The FTIR spectral data with wave number and the corresponding groups is given in Supplementary Table 2.

The stability of the evident peaks and the absence of new ones in the physical mixing and freeze-dried formulation indicate that there is physical interaction between the drug and stabilizer.²⁸

DSC and XRD

DSC assessment was accomplished to evaluate the thermal characteristics of the drug, PLGA, and the NBs pre- and post-storage for 3 months (Figure 6). The pure drug exhibited a clear endothermic peak at 167.36 °C, signifying its MP a broad peak at 287.64 °C, and at 43.31 °C, indicating its crystalline characteristics.²⁸

The thermograph of PLGA exhibited peaks at 61.8 °C and 163.17 °C. In the thermographs of the NBs, two peaks emerged at 200.52 °C and 26.18 °C, indicating the minor shifts in the MP of the drug and its confinement within the polymeric structure due to weak intermolecular interactions between the drug and the polymer.

The XRD patterns are depicted in Figure 6B. The drug has displayed firm diffraction peaks (2q scattered angles of 12.88, 16.52, 18.07, 19.65, 20.52, 21.3, 24.06, 24.2, 24.53, and 33.530) confirming its crystalline nature.^{28, 29} Previous studies have also reported similar diffraction peaks for the drug under study. However, in the NBs, the characteristic diffraction peaks of BTZ vanished, suggesting the pure drug may have formed a solid-state complex at a molecular level.

Drug release

Figure 7 depicts dissolution profiles of plain drug and drug-loaded NBs with and without acoustic assistance. Drug release from NBs was significantly higher than from a simple drug suspension. Notably, ultrasound assistance increased drug release. The cumulative drug release at 8 hours was 17.24±3.32%, 40.04±4.61%, and 76.84±4.04% for plain drug, NBs without and with acoustic activation, respectively. By 48 h, over 98.14±8.47% was released from NBs with acoustic assistance and 68.02±8.26% without acoustic aid, but in the case of the pure drug, only 33.1±4.60% of the drug was released. Drug release occurred due to cavitation collapse induced by acoustic waves and disrupted NB structures, enabling rapid medication release. Acoustic waves, characterized by meticulous control and a non-invasive nature, offer accurate drug delivery and targeting abilities. These findings confirmed that ultrasound assistance plays a pivotal role in enhancing drug release from the NBs, potentially through the cavitation effect induced by ultrasound. Ultrasound stability studies indicated the transformation of the gas core, where nanodroplets turn into bubbles, known as acoustic droplet generation. Under the influence of ultrasound, the oscillation of bubbles can trigger the shell to open, thereby aiding in the release of drugs. The study aligned with previous findings on NB stability under varying temperature conditions. The acoustic streaming flow generated by bubble oscillation regulates the movement of detached materials, which is influenced by both the radial excursion and the duration of the ultrasound pulse.¹⁸

Stability studies

BTZ-loaded NBs (NBs) underwent stability assessments at various storage conditions at 4 °C, at 25 °C, and at 40 °C immediately, one month, two months, and three months (Table 3). At 4 °C and 25 °C, minimal changes in drug content indicated robustness, with % EE showing slight variation, suggesting protection against degradation. However, a notable reduction in % EE occurred at elevated temperatures, where % EE was reduced to 62.41±3.90% from 65.12±2.54%, indicating structural disruption. Throughout the experiment, the PS of the formulation was less than 200 nm, and the ZP was around 29±2.29 mV, highlighting the stability and uniformity of BTZ NBs. Storage in a polyethylene pouch led to a faster drop in the number concentration compared to when stored in a glass bottle. Hydrogen bonding interactions were emphasized as critical factors in forming bulk NBs and their exceptional long-lasting stability.¹⁷

Pharmacokinetic studies

Figure 8 displayed the plasma concentration-time curve after drug administration in a 0.25% w/v sodium carboxymethyl cellulose solution and the optimized NBs, administered orally. Pharmacokinetic data in Table 4 revealed that the formulation exhibited significantly higher T_max, C_max (p<0.001**), AUC₀-24 (p<0.001**), and AUC₀-∞ (p<0.001**) values compared to the pure drug suspension at the prescribed dose. The bioanalytical chromatogram indicated drug retention time at 4.0 min and internal standard (Antipyrine) at 6.8 min (Supplementary Figure 3).

The optimized formulation reached a maximum level (C_max) 1.69 times higher, while the area under the curve (AUC_0-t) was 1.63 times higher than that of the free drug. In vivo studies revealed a progressive drug release from the NB preparation, with an extended half-life. The comparison of the data to the free drug shows that oral bioavailability has significantly improved. These findings suggested a notable improvement in oral bioavailability compared to the free drug. The enhanced bioavailability can be attributed to the increased drug circulation at the nanoscale and the improved penetration facilitated by the polymeric carrier system.

DISCUSSION

In this study, BTZ-loaded PLGA NBs were formulated using the solvent evaporation method, and optimization was conducted through the BBD. NBs are emerging as a formulation strategy because of their targeting ability. The process in which nanodroplets in perfluoro pentane transition from liquid to vapor upon exposure to ultrasound waves is called acoustic droplet vaporization.²⁴ Perfluoropentane undergoes a phase transition that turns nanodroplets into NBs with high US wave reflectivity. The echogenic qualities of perfluoropentane make it visible in ultrasonography images.²³

The NBs were developed using PLGA polymer containing unbound carboxylic end chains, with perfluoropentane for the inner core and PLGA as the outer shell.²³ The term “ultrasound” describes mechanical vibrations or pressure waves that exhibit compressional and rarefactional pressure fluctuations and have frequencies equivalent to or higher than the human hearing threshold (20 kHz). Ultrasound effects primarily involve two mechanisms: cavitation and sonoporation. The cavitation effect plays a crucial role in reducing the size of bubbles, whereas the sonoporation effect facilitates the uptake of these reduced bubbles.³⁰ Hydrophobic interactions play a crucial role in bonding PLGA with the drug by forming NBs, while polyvinyl alcohol (PVA) acts as a stabilizing agent. PVA creates a protective coating around the NBs to uphold their stability. Due to its biocompatibility and biodegradability properties.

Combining ultrasound with NBs helps in drug localization while overcoming off-target effects. NBs utilizing PLGA have garnered attention for targeted drug delivery because of their unique physical and surface properties. PLGA is highly recommended in various medical applications, including sutures, bone implants, and sustained drug release systems, due to its biocompatibility and biodegradability properties.¹⁴ The quadratic model suggested by the design was applied to PS, PdI, ZP, and % EE. Positive coefficients in the model indicated that an increase in associated variables led to higher drug entrapment. The concentration of the stabilizer has influenced PS, PdI, and ZP. However, when it came to % EE, a high stabilizer concentration led to a decrease in drug entrapment. PVA, as a stabilizer, has the potential to impede drug entrapment in NBs due to its competition for surface adsorption and the subsequent increase in solution viscosity. Moreover, PVA may hinder drug diffusion into NBs during the formation process. Furthermore, its destabilizing effects can result in premature rupture or aggregation, ultimately diminishing the efficiency of drug entrapment.³¹ FTIR studies confirmed compatibility between the drug and excipients. DSC and XRD studies revealed no distinct drug peak in the formulation, indicating the absence of crystalline drug material.^{10, 28}

Electron microscopy helped in the direct visualization of NBs, assessment of their integrity, and analysis of their gas composition. SEM analysis displayed homogeneous, smooth, spherical NBs. Drug release occurred due to cavitation collapse induced by acoustic waves, disrupting NB structures and enabling rapid delivery of medication. Ultrasound stability studies indicated the transformation of the gas core from nanodroplets to bubbles, known as acoustic droplet generation.³² The study aligned with the previous findings on NB stability under varying temperature conditions. The temperature-dependent behaviour observed in PS, ZP, and entrapment emphasizes understanding NB characteristics in diverse environmental conditions for practical drug delivery applications. Different polymer materials submerged in NB dispersions exhibited varied effects on NB number concentrations due to hydrophobic interactions. In vivo studies in Wistar rats revealed gradual drug release from the formulation, leading to an increased half-life of the drug with high AUC. These findings indicated a significant improvement in the oral bioavailability of the chosen medicine when using NBs compared to the free drug.³² The improved bioavailability of NBs with PLGA is achieved through various mechanisms. These include better drug encapsulation within PLGA nanoparticles, longer circulation time due to the protective coating of PLGA, and increased uptake by target cells or tissue. As a result, these mechanisms contribute to enhanced therapeutic outcomes. By enhancing tumor penetration and drug release, US-driven cavitation helps to precisely and site-specifically activate drugs. By combining ultrasound with NBs, drug localization can be achieved while mitigating off-target side effects.Optimizing stability, dose, and safety remains difficult, even if it could help reduce side effects and counteract medication resistance. Clinical acceptance depends on standardizing US characteristics among different modalities. Rigorous trials, translational research, and regulatory validation collectively define the successful incorporation of treatments. To guarantee scalability, large-scale manufacturing has to be cost-effective and efficient. Promoting this technology requires a multidisciplinary strategy to link actual uses with experimental results.

CONCLUSION

This research introduced an innovative approach to improving the solubility of BTZ NBs. By conducting a thorough investigation, the study demonstrated that NBs significantly enhanced the release of the drug, indicating their potential as a new and smart delivery system. Response surface methodology ensured a precise control over the size distribution, resulting in improved uniformity. Additionally, drug-loaded NBs exhibited exceptional stability and dissolvability in the gastrointestinal tract compared to the traditional drug suspensions, suggesting a longer drug half-life and increased effectiveness. These findings highlighted a promising role of PLGA NBs in ultrasound-responsive formulations for cancer therapy, offering notable benefits such as faster dissolution rates, sustained, targeted drug release, and improved oral bioavailability.