The physical mixtures of drug (paclitaxel) and polymer were prepared (1:1 w/w) with different excipients such as poloxamer (0.5, 1, 1.5, and 2%) and SLS (0.5%). The samples were prepared by simple mixing of drug, polymer, and excipients. The samples were stored for 1 month at 40° ± 2°C and 75 ± 5% RH (Peça et al., 2012). These physical samples were analyzed by FTIR for drug, polymer, and excipients preformulation compatibilities in comparison with nanoformulations.

The interactions between drug (paclitaxel), polymer, and excipients were carried out by preparing binary mixtures. Drug content, physical consistency, and FTIR spectra were examined at each sampling point for any possible drug–excipient incompatibility. The physical interactions among the excipients, drug, and polymer were observed by noting changes in physical consistency.

The samples containing excipients, excipients and drug, and excipients and polymer were stored under stress conditions and analyzed for determination of the drug content. Samples and standard solutions were dissolved in acetonitrile (ACN) for analysis. The drug content was measured in triplicate.

An FTIR spectrophotometer was used to analyze the samples for incompatibilities. The samples were prepared by the potassium bromide (KBr) pellet method. Dried potassium bromide was mixed with 1% w/w of the sample and grounded for 3–5 min. The sample was pulverized and converted to a compact mass by compression. The samples were analyzed in the region of 400–4,000 cm⁻¹.

The formulations were evaluated for size, polydispersity index (PDI), and zeta potential by dynamic light scattering (DLS, at 90° angle and 25°C) using a zetasizer (ZS-90, Malvern Instruments and Malvern, United Kingdom). The surface charge can be determined through zeta potential, i.e., the movements of charged particles in an electric field to predict the stability of colloids. The sample (0.5 ml) of nanoformulation and 1 ml of distilled water were taken, sonicated for 2 min, and placed in cuvettes. An average of three reported values was taken using Malvern software and analyzed statistically (Marsalek, 2014).

Drug loading efficiency (%, w/w) and drug encapsulation efficiency (%, w/w) of paclitaxel in nanoformulations were determined by centrifugation (15,000 rpm at 25°C for 30 min), followed by UV spectroscopy at 235 nm. The absorbance of the samples was measured, and the % drug loading and % encapsulation were determined by the following formulae (Huang et al., 2007): % DL = Weight   of   Drug   in   Nanoparticles Weight   of   Nanoparticles × 100 ; (1) % EE = Weight   of   Drug   in   Nanoparticles Weight   of   Drug   Feed × 100. (2)

The morphology of the sample was determined by SEM. The sample was prepared for SEM as per standard protocol in order to make it conducive. The sample was then analyzed for its morphology.

An X-Ray diffractometer (JDX-3532, Jeol, Japan) was used to carry out XRD patterns of paclitaxel, PLGA, Poloxamer 407, SLS, and paclitaxel nanoformulations. The XRD pattern was determined for its amorphous, semicrystalline, and crystalline nature. The pattern was taken at 3°–40° (2θ).

The dialysis diffusion method was applied for release studies. The membrane having a molecular weight 12,000–14,000 Da was cut in such a way that it can accommodate 2 ml redispersed nanoformulations sealed at both ends. It was then dialyzed against 100 ml of PBS (pH 7.4) in a shaking water bath at 37°C and 60 rpm. At specified time intervals (0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, 36, 48, 72, and 96 h), 2 ml sample was withdrawn and analyzed for drug release. An equal volume of dialyzing media was replaced for each sample. The drug content was determined by using a UV spectrophotometer at 235 nm in each sample. The analysis was conducted in triplicate (Bernkop-Schnürch & Jalil, 2018).

The drug release mechanisms were evaluated by applying various release kinetic models (Paarakh et al., 2018).

In Vitro cytotoxicity assay of paclitaxel-loaded polymeric nanoparticles and paclitaxel-loaded polymeric nanoparticles conjugated with trastuzumab antibody and Taxol^® was conducted by MTT [yellow tetrazolium salt, 3-(4, 5-dimethylthizol-2-yl)-2, 5, 5-diphenyl tetrazolium bromide] assay using MCF-7 breast cancer cell lines, a widely studied epithelial cancer cell line that has characteristics of differentiated mammary epithelium derived from breast adenocarcinoma (Lee et al., 2015). MCF-7 cell lines of breast adenocarcinoma show moderate overexpression of HER⁺² and serve as an excellent model for in vitro cytotoxic studies (Dhiman et al., 2004). Its hormone sensitivity through expression of estrogen receptor makes it an ideal model for in vivo and in vitro studies (Holliday & Speirs, 2011). The cells were seeded in a 96-well plate at a density of 1.0 × 10⁴ cells/well and incubated for 24 h at 37°C in 5% CO₂ at an 85% humidity incubator (Model NU 5700; United States). The medium was replaced after 24 h by paclitaxel-loaded polymeric nanoparticles and paclitaxel-loaded polymeric nanoparticles conjugated with trastuzumab and Taxol^® at concentrations ranging from 0.25 μg/ml to 50 μg/ml for 24, 48, and 72 h at 37°C. At specific intervals, the formulations were removed, and 5 mg/ml MTT was added before incubation for 4 h at 37°C. The culture solution was aspirated, and the resulting formazan crystals were dissolved in 100 µl of dimethyl sulfoxide, and the absorbance was measured at 570 nm using a microplate reader (Model FL ×800; Biotek, Winooski, VA, United States). Cytotoxicity was expressed as percentage of cell viability compared to untreated control cells. % Viability = Absorbance of sample Absorbance of control × 100. (3)

The New Zealand rabbits weighing 1.5–2.0 kg were purchased from the NIH (National Institute of Health), for in vivo pharmacokinetics. The design and study was approved by the Ethical Committee of Pharmacy Department, University of Swabi (Pharm/EC/002). The rabbits were given access to water and food. The animals were excluded by killing/using chloroform anesthesia during the study in case of any distress. The dose at the rate of 2 mg/kg body weight was injected into the marginal ear vein of rabbits, which were divided into two groups for the paclitaxel test and reference formulations. At designated time intervals (10 min, 30 min and 1, 2, 4, 6, 8, 12, 24, 96, and 120 h), blood samples were collected in EDTA tubes and centrifuged at 8,000 rpm for 10 min at 4°C. The Eppendorf tubes were used to collect and store samples at −20°C till analysis. HPLC-UV was used for the analysis of samples.

Various pharmacokinetic parameters such as peak plasma concentration (C_max), time of peak plasma concentration (T_max), elimination rate constant (K_el), elimination half-life (t_1/2), area under the plasma concentration-versus-time curve (AUC_0-∞), clearance (Cl), steady state volume (V_ss), and mean residence time (MRT) were determined using PK-Summit^® software.

For the quantification of paclitaxel in samples, mean (X), SD, and %RSD were applied. Comparison between means of treatments was made at p ≤ 0.05 using the Student t test.

The samples were prepared using binary mixtures of the drug, polymer, and excipients (1:1), stored for 01 months under stress conditions, and inspected visually for any change in color and texture. The drug, polymer, and excipients compatibility study was performed by FTIR at day 1 and 30. The results of FTIR are shown in Table 2 and Figure 1. The concentration of the standard drug and samples at day 1, 15, and 30 were evaluated as given in Table 3.

As shown in Figure 1A, the FTIR spectra of paclitaxel show characteristic peaks at 3,441 cm⁻¹ (for O-H stretching), 3,309 cm⁻¹ (for N-H stretching), aromatic C-H at 2,920–2,850 cm⁻¹, peaks at 1708 cm⁻¹ for C = O stretching vibration of the ester group, peak at 1,647 cm⁻¹ for the amide bond, and peaks at 1,254 cm⁻¹ for C-N stretching. The FTIR spectra of PLGA (75:25) showed distinct peaks at 3,200 cm⁻¹ for -OH stretching, 2,943 cm⁻¹ for -CH stretching, 1751 cm⁻¹ for carbonyl –C = O stretching, and at 1,072 cm⁻¹ for C-O stretching as given in Figure 2B. The FTIR spectra of poloxamer 407 showed characteristic peaks at 1,111 cm⁻¹ and 1,060 cm⁻¹ distinguishing of its PEO group and at 2,881 cm⁻¹ for CH₂-CH₂ stretching as shown in Figure 1C. The FTIR spectra of SLS showed characteristic peaks at 1,219–1,153 cm⁻¹ for S-O stretching and at 2,850 cm⁻¹ for -CH stretching as shown in Figure 1D.

The characteristic peaks of paclitaxel was not present in the FTIR spectrum which means that the drug is completely encapsulated by the polymer, but the main peaks for PLGA, poloxamer 407, and SLS remain the same indicating the absence of any interaction between the drug, polymer, and stabilizers used as shown in Figure 1E.

The particle size was within the range of 180 ± 1.22 to 202 ± 36.17 nm for 0.5%, 199 ± 21.80 to 224 ± 26.98 nm for 1%, 202.3 ± 14.5 to 224 ± 26.98 nm for 1.5%, and 229 ± 13.24 to 408 ± 11.27 nm for 2% poloxamer 407. The particle size, PDI, zeta potential, and drug loading and encapsulation efficiency of paclitaxel nanoparticles are given in Table 4. The physicochemical properties and encapsulation efficiency of paclitaxel nanoparticles with or without antibody conjugation are given in Table 5.

SEM was used for determining surface morphology of simple paclitaxel nanoparticles and conjugated paclitaxel nanoparticles as shown in Figures 2A,B and Figures 2C,D, respectively.

The XRD patterns of paclitaxel, PLGA, Poloxamer 407, SLS, and paclitaxel nanoformulations are shown in Figure 3A, in which paclitaxel exhibits several peaks at 2θ value of 5.4°, 8.8°, and 12.25° which shows the crystalline nature of paclitaxel, while no peaks were observed for PLGA which depicts the amorphous nature of the polymer as shown in Figure 3B. The diffractogram of SLS shows two distinct peaks at 2θ value of 20.3° and 21.65° demonstrating the crystalline nature of SLS (Figure 3C), while poloxamer 407 exhibits peaks in the 2θ range at 19.05° and 23.2° as shown in Figure 3D). The XRD pattern of paclitaxel-loaded PLGA nanoformulations exhibits no discrete peaks at any position (Figure 3E).

The in vitro release profile of paclitaxel nanoformulations and surface-modified nanoformulations was determined. At specified time intervals (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216, 240, and 264 h), the samples were withdrawn and analyzed for drug release. All the paclitaxel-loaded PLGA nanoformulations and surface-modified nanoformulations exhibit a bi-phasic release pattern as shown in Figures 5, 6, respectively, which is characterized by an initial burst release in first 24 h followed by a continuous slow release. The initial burst release of paclitaxel from nanoformulations at 24 h was 26 ± 0.23 and 28 ± 0.42% for PTX 108 and PTX 112, while at 264 h drug release was 79 ± 0.09 and 81 ± 0.43% for PTX 108 and PTX 112, respectively. Similarly the initial burst release of paclitaxel from modified nanoformulations at 24 h was 30 ± 0.28 and 33 ± 0.03% for PTX 108ab and PTX 112 ab, while at 264 h, the drug release was 85 ± 0.34 and 88 ± 0.14% for PTX 108 ab and PTX 112 ab, respectively.

Various kinetic models were employed for prediction of drug release mechanisms that include zero-order, first-order, Hixson–Crowell, Korsmeyer–Peppas, and Higuchi. The regression coefficient values (R ²) obtained and the drug release from PTX 108, PTX 112, PTX 108ab, and PTX 112 ab nanoformulations best fit to the Higuchi model on the basis of higher regression. coefficient (R ²) values as shown in Table 6.

The cytotoxicity of the reference drug and paclitaxel-loaded PLGA nanoparticles with and without antibody surface modification was evaluated by performing MTT assay against MCF-7 cells. The MCF-7 cell lines were incubated with Paclixil^®, paclitaxel-loaded PLGA nanoformulations PTX 112, and antibody-conjugated paclitaxel-loaded PLGA nanoformulations PTX 108ab and PTX 112 ab at 0.25, 2.5, 10, 25, and 50 μg/ml concentration. The cultured cells were analyzed for cell viability at 24, 48, and 72 h. Cytotoxicity as % of cell viability compared to untreated control cells is shown in Figure 7.

Rabbits weighing between 1.5 and 2 kg were used as an experimental model for the assessment of various in vivo pharmacokinetic parameters of the selected paclitaxel nanoformulations. Selected paclitaxel and its commercially available paclitaxel formulation Paclixil^® (reference formulation) were administered (2 mg/kg body weight) via the marginal ear vein. For in vivo evaluation of selected and commercially available paclitaxel formulation Paclixil^®, the developed RP-HPLC-UV method was successfully applied (Sakhi et al., 2021). The data were evaluated by non-compartmental analysis using PK-Summit^®. The results are given in Table 7 and Figure 8.

The stabilizers used for preparing nanoformulations may interact with each other and other active pharmaceutical ingredients which may affect the stability of nanoparticles. Due to change in temperature and humidity, the physical and chemical changes in the dosage form are occurred which can affect stability, biocompatibility, and therapeutic properties of the drug (Chadha & Bhandari, 2014; Patel, et al., 2015). In order to avoid these possible interactions, the drug, polymer, and excipients compatibility study was performed, and the samples were evaluated for drug content, physical consistency, and FTIR spectra. Drug concentration in a dosage form may decrease due to degradation of the drug when stored under stress conditions. Physical and chemical incompatibilities may be triggered by humidity and temperature. The drug contents in the dosage form remained the same throughout the stored period. The IR spectra show no changes in samples at day 1 while showing chemical interaction between drug, polymer, and other drug excipients used in nanoformulations after 30 days (Martins et al., 2014). The FTIR spectrum of the paclitaxel-loaded PLGA nanoparticle showed no characteristic peaks of paclitaxel which means that the drug is completely encapsulated by the polymer, but the main peaks for PLGA, poloxamer 407, and SLS remain same, thus indicating the absence of any interaction between the drug, polymer, and stabilizers used. After visual inspection of samples, no changes in color or physical consistency were noted which indicates the compatibility of drugs and active ingredients with each other.

The particle size and PDI change with change in concentration of the stabilizer and amount of the drug in nanoparticle formulations, whereas polymer concentration is kept constant. The size of the nanoparticles increases as the concentration of poloxamer 407 is increased, and there is an increase in PDI with change in stabilizer concentration. Present studies show that the mean particle size increases as the concentration of the stabilizer is increased (Pradhan et al., 2013). This increase in the nanoparticle size is due to excessive adsorption of poloxamer 407 on the nanoparticle surface which results in formation of a thick layer (Redhead et al., 2001). As the stabilizer concentration is increased, viscosity of the aqueous phase increases which results in an increase in particle size by decreasing the net shear stress (Pradhan et al., 2013). As the drug concentration is increased from 1 to 4 mg, there is an increase in the nanoparticle size. This increase in particle size is due to the fact that only a specified amount of the drug can be encapsulated by a constant concentration of the polymer. Any further increase in drug concentration will result in an increase in particle size, thus increasing viscosity of the organic phase (Mu & Feng, 2003; Pradhan et al., 2013). The results show that after the attachment of trastuzumab on the nanoparticle surface there is an increase in size and polydispersity of nanoparticles.

Paclitaxel-loaded polymeric nanoformulations prepared by using poloxamer 407 (0.5, 1, 1.5, and 2%) and SLS (0.05%) show a negative charge, and the zeta potential values decrease as the concentration of the drug is increased from 1 to 4 mg. This decrease in zeta potential is due to an increase in concentration of the drug-to-polymer ratio in the organic phase (Stolnik et al., 2001; Moura et al., 2020). The negative zeta potential was due to the ester and termination group of PLGA chains on the nanoparticle surface (Mu & Feng, 2003) and due to the presence of the anionic surfactant, SLS. As the concentration of poloxamer 407 is increased, there is an increase in zeta potential values. There is an increase in zeta potential values ranging from −19.1 ± 1.5 to −40.4 ± 1.6 mV as the poloxamer 407 concentration is increased from 0.5 to 2% (Redhead et al., 2001; Reddy & Murthy, 2005). High negative potential provides stability as there will be an increase in electrostatic repulsive forces among the nanoparticles which will prevent particle aggregation. The results indicated that the surface charge was shifted to less negative after conjugation of the antibody on the nanoparticle surface due to the positive charge of trastuzumab (Sun et al., 2011a; Sun et al., 2008; P.; Yousefpour et al., 2011).

There is an increase in encapsulation efficiency as the concentration of poloxamer 407 is increased from 0.5 to 2% while keeping PLGA and SLS concentrations constant (Table 4). There is an increase in encapsulation efficiency as the initial concentration of the drug is increased, as more drug molecules are available to interact with the polymer resulting in an increase in encapsulation efficiency. However any further increase in the drug amount will result in saturation of the polymer, leading to a decrease in encapsulation efficiency (Keum et al., 2011; Pradhan et al., 2013). In this work, nanoformulations having encapsulation efficiencies greater than 80% were selected for surface modification which resulted in an increase in size; however, no significant change in encapsulation efficiency was observed with conjugation of the antibody as shown in Table 5.

Surface morphology of nanoparticles determines the circulation time, biodistribution, targeted delivery, and enhanced tumor accumulation as well as cellular uptake of nanoparticles (Truong et al., 2015). The surface of nanoparticles using poloxamer 407 was spherical in shape. After the conjugation of the antibody, the surface of nanoparticles becomes blurry which is due to attachment of the antibody on the surface of nanoparticles and adhesion of nanoparticles (Yousefpour et al., 2011; Mehata et al., 2019).

The XRD pattern of paclitaxel-loaded PLGA nanoformulations exhibits no discrete peaks at any position, so it can be concluded that paclitaxel was completely encapsulated by the polymer and transformed to an amorphous state (Chowdhury et al., 2019; de Oliveira Fortes et al., 2012; Wei et al., 2009).