Particle Size Analysis and Reduction Techniques to Enhance Drug Bioavailability in Preclinical Studies

Transforming new chemical entities (NCEs) into effective drug molecules is a complex and challenging task ^[1], especially for poorly soluble BCS class II/IV drugs ^[2]. To enhance the oral bioavailability of these compounds, researchers often employ various optimization strategies aimed at improving solubility and permeability. For poorly soluble compounds, drug particle size reduction is considered one of the main strategies to improve absorption (Figure 1) ^[3].

Figure 1. Optimization strategies for BCS class II-IV drugs

Particle size directly affects the adaptability, stability, and absorption rate of drug formulations in biological systems. Smaller particles provide a larger specific surface area, which not only promotes drug particle dissolution but also enhances interaction with cell membranes. Optimization strategies for drug particle size typically include micronization, nanoparticles, micro-emulsification, and solid dispersion techniques. For compounds with low oral bioavailability due to poor solubility, particle size reduction is an effective way to improve absorption, increase bioavailability, and optimize pharmacokinetic characteristics. This article discusses the impact of particle size on oral drug absorption and summarizes different particle size reduction and analysis methods and their pros and cons in preclinical studies.

Impact of Particle Size on Oral Absorption

Oral administration is the most common route in clinical applications, with solid formulations being the predominant dosage form. Drugs taken orally undergo a series of complex processes in the gastrointestinal tract, including disintegration, release, dissolution, and transport across intestinal epithelial cells, ultimately being absorbed into the systemic circulation. The absorption of oral drugs primarily relies on passive diffusion, active transport, and facilitated diffusion, and reducing particle size can effectively enhance these absorption pathways.

Particle size reduction increases the specific surface area, facilitating both the dissolution and membrane transport processes ^[4]. This can be achieved through various techniques like micronization and nanoparticle formation. Micronization uses mechanical force to reduce particle size, while nanoparticles are prepared through solvent exchange or phase separation processes. These techniques not only improve solubility but also affect drug distribution and bioavailability.

Case Studies on Particle Size Reduction: Nanoparticles vs. Micronization

Wu Y et al. found that a 0.12 µm formulation of aprepitant achieved a C_max four times higher than a 5.5 µm formulation in beagle dogs, indicating that appropriate micronization can enhance drug absorption and bioavailability.

Figure 2. Plasma concentration profiles of aprepitant suspensions with different particle sizes

Alshora D et al. demonstrated that rosuvastatin calcium nanoparticles achieved twice the C_max and 1.5 times the AUC of untreated drugs in rabbits, with significantly improved dissolution rates and plasma drug concentrations for ground nanoparticles.

Figure 3. Dissolution rate (A) and plasma concentration (B) profiles of untreated and ground nanoparticles

Nekkanti et al. found that in rats, nanoparticles of oral Candesartan cilexetil with a median size of 127 nm increased AUC by 2.5 times and C_max by 1.7 times compared to micronized suspensions, also reducing T_max from 1.81 hours to 1.06 hours.

Figure 4. Plasma concentration profiles of oral candesartan cilexetil particles with different sizes in rats

These results demonstrate a direct relationship between particle size and drug absorption, underscoring the importance of controlling particle size in drug formulation development to enhance bioavailability and therapeutic efficacy.

Particle Size Reduction Enhances Dissolution

The size of drug particles directly influences their dissolution rate in the gastrointestinal tract. Studies have shown that smaller particles result in faster disintegration and drug release, thereby accelerating the dissolution process. Researchers evaluated the dissolution rates of drugs with different particle sizes (esomeprazole, a proton pump inhibitor) using laser diffraction. As shown in Figure 5, formulations with an X₅₀ of 648 µm (Nexium) had a median dissolution time (T₅₀) of about 61 minutes, whereas formulations with an X₅₀ of 494 µm (Actavis) reduced T₅₀ to about 38 minutes. The results indicate an inverse relationship between particle size and dissolution rate; smaller particles dissolve faster. Thus, optimizing particle size can effectively regulate drug solubility and improve bioavailability ^[8].

Figure 5. Drug release in a simulated gastrointestinal pH environment with different particle sizes

Particle Size Reduction Enhances Membrane Absorption

The transport of drugs across intestinal cell membranes involves penetrating the bilayer mucus and epithelial cell membrane. As shown in Figure 6, the mucus layer is composed of high-molecular-weight mucins with pores ranging from 10 nm to 200 nm. Adjusting drug particle size within this range can extend residence time in the mucus layer, enhancing penetration through the intestinal wall. Previous studies have shown that drugs with particle sizes below 200 nm can smoothly traverse the bilayer mucus, enter epithelial cells, and be absorbed into the blood or lymph, reaching target tissues and organs ^[9-11].

Figure 6. Schematic diagram of the small intestine structure

As shown in Figure 7, drug absorption in the intestine primarily involves persorption, transcellular uptake, and paracellular uptake. Studies have indicated that particle size is closely related to the efficiency of these absorption pathways ^[12]. However, conflicting results exist regarding the absorption of drugs with particle sizes of 50 nm and 100 nm, although it is generally believed that drugs within the 50-100 nm range are more easily absorbed by the intestine ^[13-16].

Figure 7. Transmembrane transport modes of intestinal epithelial cells

Comparison of Particle Size Reduction Techniques

In drug formulation development, particle size reduction is an effective strategy to enhance the bioavailability of poorly soluble drugs, applicable to both solid and liquid formulations. Various optimization strategies and methods are employed based on formulation characteristics and requirements, as shown in Table 1 ^{[12, 17]}.

Table 1. Comparison of particle size reduction techniques

In preclinical formulation preparation, homogenization techniques are often used for particle size reduction. Conventional homogenization techniques frequently employ ultrasonication, and high-power focused ultrasonication can effectively reduce particle size. Combining liquid antisolvent crystallization with focused ultrasonication, one can select antisolvents based on structural formulas or screening experiments, effectively enhancing drug absorption in the intestine (as shown in Table 2).

Table 2. Comparison of particle size reduction limits for different techniques

In summary, particle size reduction techniques play a crucial role in enhancing drug absorption. When choosing a particle size reduction technique, factors such as drug characteristics, desired particle size range, and economic and technical considerations must be considered.

Comparison of Particle Size Analysis Techniques

Accurate particle size analysis is essential. Current particle size detection techniques are mainly divided into flow scanning and field scanning categories. These techniques capture photoelectric signals related to particle shape and use mathematical models to convert these signals into statistical mean values, quantitatively describing particle dimensions. However, differences may occur between instruments due to equivalent particle size, particle shape, and particle dispersion, leading to variations in particle size data. Therefore, interpreting particle size data requires detailed data analysis and appropriate mathematical descriptions ^[18].

Mainstream Particle Size Detection Techniques ^[19]

Laser Diffraction (LD) is widely used due to its fast measurement without the need for centrifugation. LD accurately measures particle size distribution but assumes spherical particles, which may lead to significant bias for non-spherical particles. Additionally, sample dilution is generally required to obtain accurate results, possibly affecting particle interactions, especially with complex samples.

Dynamic Light Scattering (DLS) is suitable for analyzing complex samples, including non-spherical and self-aggregating particles, providing information about particle size and shape, and handling higher sample concentrations. However, DLS is relatively complex to operate, usually requiring centrifugation, and its results also assume spherical particles, which may lead to measurement bias for non-spherical particles and limit concentration analysis of non-self-aggregating particles.

Scanning Electron Microscopy (SEM) allows direct observation of particle size and shape without sample dilution. Although SEM provides high-resolution images, it is cumbersome to operate and not suitable for large-scale sample processing. The limited field of view may increase statistical errors, and distinguishing overlapping particles is challenging.

Transmission Electron Microscopy (TEM) provides three-dimensional information for overlapping particles, and is suitable for direct observation of particle size and shape. However, TEM operation is also cumbersome, not suitable for large-scale sample processing, and may result in significant statistical errors.

Comparison of Detection Ranges for Mainstream Particle Size Analysis Techniques

The comparison of the detection ranges for the above particle size detection techniques is summarized in Figure 8.

Figure 8. Comparison of detection ranges for mainstream particle size analysis techniques

In summary, different particle size detection techniques have different advantages and limitations. Selecting the appropriate technique requires considering sample characteristics, analysis objectives, and experimental conditions. In drug formulation development, the comprehensive use of multiple techniques to improve analysis accuracy and reliability is crucial.

Case Study of Particle Size Reduction and Determination Techniques

In this experiment, we selected the commercial compound Cinnarizine for preparation, aiming to achieve a formulation with particle sizes below 200 nm. The solvent-antisolvent precipitation method was used, and a focused ultrasonication device (Covaris) was employed for particle size reduction. The treated particle size was subsequently measured using a Malvern analyzer, achieving the expected nanoscale formulation. As shown in Figure 9, the particle size distribution range after ultrasonication ranged from 10 nm and 10,000 nm, with a median particle size (X₅₀) of approximately 200 nm, successfully reducing the particle size into the expected range.

Figure 9. Particle size distribution in validation experiments

The experiment used Covaris's focused ultrasonication device (as shown in Figure 10, left) for particle size reduction. This device utilizes Covaris's Adaptive Focused Acoustics (AFA) technology, providing precise adaptive acoustic energy control. During the experiment, the device precisely controlled cavitation and acoustic streaming within the sample processing container, generating and transmitting focused acoustic energy to individual samples. This process led to a series of controlled compression and rarefaction events, achieving non-contact, isothermal acoustic processing of the sample without damaging it. The experimental parameters are shown in Table 3.

Table 3. Experimental conditions for nanoparticle preparation

The particle size analysis was conducted using the Mastersizer 3000 laser particle size analyzer (Figure 10, right). This instrument employs a patented folded optical path design and coaxial dual light source technology, enabling detection of particles ranging from 10 nm to 3.5 mm. The instrument demonstrated verifiable accuracy and repeatability, exceeding the recommended standards of ISO 13320:2009 and USP <429>.

Figure 10. Covaris ultrasonicator (left) and Mastersizer particle size analyzer (right)

Concluding Remarks

Particle size reduction can significantly improve the poor gastrointestinal absorption of poorly soluble drugs. Numerous established methods can effectively reduce compound particle size. In preclinical studies, due to the limited amount of compound and formulation volume, focused ultrasonication combined with laser diffraction detection analysis can efficiently achieve particle size reduction and rapid distribution measurement, making them relatively optimal experimental methods for preclinical evaluation. Moreover, particle size research is closely linked with drug efficacy and safety, and is an indispensable part of drug formulation development. It is crucial for accelerating new drug development and enhancing drug quality. WuXi AppTec DMPK has comprehensive preclinical compound particle size reduction and detection technologies, combined with preclinical in vivo pharmacokinetic experiments, to evaluate the impact of particle size reduction on in vivo bioavailability and pharmacokinetic characteristics, aiding drug development progress.

Authors: Yuan Wen, Quanli Feng, Furong Jiao, Cheng Tang

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