A Review on Nanoparticles: Synthesis, Characterisation, and Applications in Pharmaceutical Sciences

Figure 1. Historical evolution of nanoparticle-based drug delivery systems from conventional carrier systems to advanced intelligent nanomedicines.

1.3 Significance and Advantages in Pharmaceutical Sciences

The incorporation of nanoparticles into pharmaceutical formulations offers numerous compelling advantages that address critical limitations of conventional drug delivery approaches:

• Enhanced Solubility and Bioavailability: Many promising therapeutic agents, particularly anticancer drugs and natural compounds, suffer from poor aqueous solubility, leading to low oral bioavailability and requiring high doses or parenteral administration. Nanoparticle encapsulation can dramatically increase the apparent solubility of hydrophobic drugs, with some systems achieving up to 100-fold improvement in dissolution rates compared to conventional formulations.
• Targeted Drug Delivery: Nanoparticles can be engineered to selectively accumulate in target tissues through passive targeting (EPR effect in tumors) or active targeting (ligand-receptor interactions). This targeting capability reduces systemic exposure and minimizes off-target toxicity, particularly important for cytotoxic chemotherapeutic agents.
• Controlled and Sustained Release: The drug release profile from nanoparticles can be precisely tuned through selection of carrier materials, particle size, surface modifications, and incorporation of stimuli-responsive elements. This enables sustained therapeutic levels over extended periods, reducing dosing frequency and improving patient compliance. Protection of Labile Drugs: Encapsulation within nanoparticles protects sensitive therapeutic agents from degradation by enzymes, pH changes, and other environmental factors during transit to the target site. This is particularly valuable for proteins, peptides, and nucleic acids that are rapidly degraded in biological fluids.
• Crossing Biological Barriers: The nanoscale dimensions enable nanoparticles to cross biological barriers that are impermeable to larger drug molecules. This includes the blood-brain barrier for neurological disorders, the intestinal epithelium for oral delivery of macromolecules, and the skin barrier for transdermal delivery.
• Combination Therapy: Nanoparticles can simultaneously encapsulate multiple therapeutic agents with different physicochemical properties, enabling synergistic combination therapies. This approach is particularly valuable in cancer treatment where drug resistance is a major challenge.
• Imaging and Diagnostics: Nanoparticles can be loaded with imaging agents (contrast agents for MRI, fluorescent probes, radionuclides) to enable real-time tracking of drug distribution, monitoring of therapeutic response, and early detection of diseases.^[4,5]

1.4 Classification of Pharmaceutical Nanoparticles

Pharmaceutical nanoparticles can be classified based on their composition, structure, and origin:

A. Organic Nanoparticles:

1. Lipid-based: Liposomes, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), lipid nanoparticles (LNP)
2. Polymeric: Poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), chitosan, alginate, gelatin nanoparticles
3. Dendrimers: Polyamidoamine (PAMAM), poly(propylene imine) (PPI), poly(lysine) dendrimers
4. Micelles: Polymeric micelles, surfactant micelles

B. Inorganic Nanoparticles:

1. Metallic: Gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), iron oxide nanoparticles (IONPs), copper nanoparticles
2. Metal oxide: Zinc oxide (ZnO), titanium dioxide (TiO2), cerium oxide (CeO2) nanoparticles
3. Silica: Mesoporous silica nanoparticles (MSN), silica nanospheres
4. Carbon-based: Carbon nanotubes, graphene, fullerenes, carbon dots

C. Hybrid Nanoparticles:

1.  Polymer-lipid hybrid nanoparticles
2. Metal-polymer nanocomposites
3.  Core-shell structures
4. Layer-by-layer assembled nanoparticles

4. Biological Nanoparticles:

1. Exosomes and extracellular vesicles
2. Virus-like particles
3. Protein-based nanoparticles (albumin, ferritin)
4. Cell membrane-coated nanoparticles. ^[6,7]

2. SYNTHESIS OF NANOPARTICLES

2.1 Top-Down Approaches

2.1.1 Ball Milling

Ball milling is a mechanical attrition technique where bulk material is ground in a rotating cylindrical container filled with milling media (balls) under controlled conditions. The impact and shear forces generated during milling break down the material into nanoparticles. Parameters such as milling speed, duration, ball-to-powder ratio, and use of surfactants or stabilizers significantly influence the particle size and morphology. High-energy ball milling can produce nanoparticles in the range of 10-100 nm, though the size distribution is often broad. The technique is scalable and cost-effective but may introduce contamination from the milling media and lacks precise control over particle characteristics.^[8]

2.1.2 High-Pressure Homogenization

High-pressure homogenization forces a coarse suspension through a small orifice under high pressure (typically 100-2000 bar), creating intense shear, cavitation, and impact forces that reduce particle size. This technique is widely used for producing nanosuspensions of poorly water-soluble drugs. Multiple homogenization cycles are typically required to achieve the desired particle size. The method is suitable for thermolabile compounds as it operates at ambient or slightly elevated temperatures. However, the process can be energy-intensive and may require high drug concentrations.^[9]

2.1.3 Media Milling (Nanocrystal Technology)

Media milling involves grinding drug particles in a liquid medium using milling beads (typically 0.2-0.5 mm) in a stirred media mill. Stabilizers such as surfactants or polymers are added to prevent agglomeration. This technique has been successfully commercialized for producing nanocrystal formulations (e.g., Rapamune, Emend). The process can achieve particle sizes below 400 nm and is scalable for industrial production. Challenges include potential contamination from milling media and the need for extensive washing steps.^[10]

2.2 Bottom-Up Approaches

1. 1. 1. Precipitation Methods
1. Solvent Evaporation Method: In this widely used technique, the drug and polymer are dissolved in a volatile organic solvent (typically dichloromethane, chloroform, or ethyl acetate). The organic solution is then emulsified in an aqueous phase containing a surfactant or stabilizer using high-speed homogenization or sonication. The organic solvent is subsequently evaporated under reduced pressure or by stirring at ambient temperature, causing the polymer to precipitate around the drug and form nanoparticles. The nanoparticles are collected by centrifugation and washed to remove residual solvent and surfactant. This method is suitable for hydrophobic drugs and can achieve high encapsulation efficiency. However, the use of organic solvents raises safety concerns and requires extensive removal steps.^[11]
2. Solvent Displacement (Nanoprecipitation) Method: Also known as the interfacial deposition method, this technique involves adding a solution of drug and polymer in a water-miscible organic solvent (such as acetone or ethanol) to an aqueous phase under moderate stirring. Rapid diffusion of the organic solvent into the aqueous phase causes precipitation of the polymer and drug, forming nanoparticles. The process is spontaneous and does not require high-energy homogenization. Hydrophobic drugs can be efficiently encapsulated, and the particle size is influenced by the solvent diffusion rate, polymer concentration, and stirring speed. The main advantages include simplicity, low energy requirements, and the ability to produce small nanoparticles (typically 100-300 nm). However, the method is limited to water-miscible solvents and may result in lower encapsulation efficiency for hydrophilic drugs.^[12]
   1. 2. Emulsion-Based Methods

1. Single Emulsion (Oil-in-Water, O/W): This method is suitable for encapsulating hydrophobic drugs. The drug and polymer are dissolved in a volatile organic solvent (oil phase) and emulsified in an aqueous phase containing an emulsifier (surfactant or stabilizer) using high-speed homogenization or sonication. The organic solvent is then evaporated, and the polymer forms nanoparticles encapsulating the drug. Common emulsifiers include polyvinyl alcohol (PVA), poloxamers (Pluronic F68), and lecithin. The particle size depends on the homogenization intensity, emulsifier concentration, and viscosity of the phases. This method is versatile and widely used for producing polymeric nanoparticles.
2. Double Emulsion (Water-in-Oil-in-Water, W/O/W): The double emulsion method is employed for encapsulating both hydrophilic and hydrophobic drugs simultaneously or for hydrophilic drugs alone. In the first step, an aqueous solution of the hydrophilic drug is emulsified in an organic solution of the polymer using high-speed homogenization, forming a primary water-in-oil (W/O) emulsion. This primary emulsion is then added to a second aqueous phase containing an emulsifier and homogenized to form the W/O/W double emulsion. The organic solvent is evaporated, and the polymer precipitates to form nanoparticles with the hydrophilic drug encapsulated in the aqueous core and the hydrophobic drug (if present) in the polymer matrix. This method is particularly valuable for protein and peptide delivery but may result in lower encapsulation efficiency and broader size distribution compared to single emulsion.^[13]

2.2.3 Supercritical Fluid Technology

1. Supercritical Anti-Solvent (SAS) Method: In the SAS method, the drug and polymer are dissolved in an organic solvent and sprayed into a vessel containing a supercritical fluid (typically supercritical CO2), which acts as an anti-solvent. The rapid diffusion of the organic solvent into the supercritical fluid causes supersaturation and precipitation of the solute as nanoparticles. The supercritical fluid simultaneously extracts the organic solvent, leaving dry nanoparticles. This technique produces solvent-free nanoparticles with controlled size and morphology. The process is environmentally friendly as CO2 is non-toxic and easily removed by depressurization.^[14]
2. Rapid Expansion of Supercritical Solutions (RESS): In the RESS method, the drug is dissolved in a supercritical fluid (typically supercritical CO2) and the solution is rapidly expanded through a nozzle into a low-pressure chamber. The sudden decrease in pressure and temperature causes the solute to precipitate as nanoparticles. The particle size is controlled by the pre-expansion temperature, pressure, and nozzle design. This method is suitable for compounds with reasonable solubility in supercritical CO2 but may not be applicable to polar compounds without co-solvents.^[15]

2.2.4 Coacervation and Ionic Gelation Methods

Coacervation Method: Coacervation involves the phase separation of a polymer solution into a polymer-rich phase (coacervate) and a polymer-poor phase. In simple coacervation, a non-solvent or change in temperature causes the polymer to separate from solution. In complex coacervation, two oppositely charged polymers interact to form a complex that separates as a coacervate phase. The drug is dispersed in the polymer solution, and coacervation is induced, causing the polymer to encapsulate the drug particles. The coacervate droplets are then hardened by cross-linking, temperature change, or solvent removal. This method is particularly useful for natural polymers like gelatin, alginate, and chitosan. Ionic Gelation Method: Ionic gelation is a mild, aqueous-based method suitable for encapsulating sensitive biomolecules. In this technique, a polyelectrolyte solution (such as sodium alginate or chitosan) containing the drug is added dropwise to a solution containing multivalent counterions (such as calcium chloride for alginate or tripolyphosphate for chitosan). The electrostatic interaction between the polyelectrolyte and counterions causes gelation and formation of nanoparticles. The method is simple, does not require organic solvents or high temperatures, and is biocompatible. However, the particle size is relatively large (typically 200-1000 nm) and the drug loading may be limited.^[16]

2.3 Green Synthesis Approaches (Emerging Trend 2023-2026)

2.3.1 Plant-Mediated Green Synthesis

Plant-mediated synthesis utilizes aqueous extracts of plant parts (leaves, roots, fruits, seeds, bark) as reducing and stabilizing agents for the production of metallic nanoparticles. The plant extracts contain a rich diversity of phytochemicals including polyphenols, flavonoids, terpenoids, alkaloids, and proteins that serve as both reducing agents (converting metal ions to metal nanoparticles) and capping agents (stabilizing the formed nanoparticles and preventing aggregation).

1. Mechanism of Plant-Mediated Synthesis: The synthesis process typically involves mixing an aqueous solution of metal salt (such as silver nitrate, chloroauric acid) with the plant extract at room temperature or mild heating. The phytochemicals in the extract donate electrons to reduce metal ions (M+) to metal atoms (M0). The metal atoms nucleate and grow into nanoparticles, while biomolecules from the plant extract adsorb onto the nanoparticle surface, acting as capping agents that prevent aggregation and provide stability. The color change of the reaction mixture (from colorless to brown for silver nanoparticles, or to ruby red for gold nanoparticles) indicates the formation of nanoparticles due to surface plasmon resonance.

Recent studies have demonstrated the successful green synthesis of gold and silver nanoparticles using crude extracts of Aconitum violaceum, a medicinal plant from the Himalayan region. The synthesized AuNPs and AgNPs exhibited spherical and triangular morphologies with average sizes below 100 nm. The phytochemical screening revealed the presence of alkaloids, flavonoids, terpenoids, saponins, and anthraquinones in the extract, with alkaloids and flavonoids identified as the primary reducing and capping agents. The green-synthesized nanoparticles demonstrated effective antibacterial activities with minimum inhibitory concentrations (MICs) of 95 and 70 microg/mL against Lactobacillus acidophilus and 90 and 65 microg/mL against Escherichia coli for AuNPs and AgNPs, respectively. The enhanced antibacterial activity compared to the crude extract was attributed to the synergistic effect of phytochemicals and the nanoparticles themselves.

2. Advantages of Plant-Mediated Synthesis: Environmentally friendly and sustainable, utilizing renewable plant resources; Elimination of toxic chemical reducing agents (such as sodium borohydride, hydrazine); Cost-effective and easily scalable; Biocompatibility due to natural capping by biomolecules; One-pot synthesis under mild conditions (ambient temperature, atmospheric pressure); Reduced energy requirements compared to physical methods.^[17,18]

Figure 2. Green Synthesis of Nanoparticles Using Plant Extracts

2.3.2 Microbial-Mediated Green Synthesis

Microorganisms including bacteria, fungi, actinomycetes, and algae have been exploited for the biosynthesis of nanoparticles. The mechanism involves enzymatic reduction of metal ions by microbial cellular components or secreted biomolecules. Intracellular synthesis occurs within the microbial cell, while extracellular synthesis utilizes enzymes or metabolites secreted into the culture medium.

1. Bacterial Synthesis:

Bacteria such as Pseudomonas, Bacillus, and Escherichia coli can synthesize nanoparticles through enzymatic reduction of metal ions. This method offers rapid growth, easy cultivation, and scalability, although purification is required to remove endotoxins.

2. Fungal Synthesis:

Fungi including Aspergillus, Fusarium, and Penicillium species are widely used for nanoparticle synthesis due to their high enzyme production and metal tolerance. Fungal synthesis often provides higher yields and facilitates easier downstream processing.

3. Algal Synthesis:

Marine and freshwater algae can produce nanoparticles using naturally occurring polysaccharides, proteins, and pigments as reducing agents. This eco-friendly approach generates biocompatible nanoparticles suitable for pharmaceutical and biomedical applications.

2.3.3 Biopolymer-Mediated Synthesis

Biopolymers such as chitosan, alginate, starch, and cellulose derivatives can serve as both reducing and stabilizing agents for nanoparticle synthesis. Chitosan, a cationic polysaccharide derived from chitin, has been extensively studied for silver and gold nanoparticle synthesis due to its biocompatibility, antimicrobial properties, and ability to form complexes with metal ions. The amino and hydroxyl groups of chitosan participate in the reduction of metal ions and stabilization of nanoparticles through coordination and electrostatic interactions.^[19,20]

2.4 Comparison of Synthesis Methods

The selection of an appropriate synthesis method depends on various factors including the nature of the drug, desired particle characteristics, scale of production, regulatory requirements, and cost considerations. Table 1 presents a comprehensive comparison of the major synthesis methods for pharmaceutical nanoparticles.^[21]

Table No. 1: Comparison of all Synthesis Methods

2.5 Recent Advances in Synthesis (2023-2026)

2.5.1 Microfluidic Synthesis

Microfluidic technology has emerged as a powerful platform for controlled nanoparticle synthesis, offering precise control over mixing, reaction conditions, and residence time. The laminar flow regime in microchannels enables rapid and uniform mixing of reagents, leading to narrow particle size distributions. Microfluidic devices can be designed with various geometries (T-junction, Y-junction, flow-focusing) to control the mixing pattern and nanoparticle formation. This technology is particularly valuable for the production of lipid nanoparticles for mRNA delivery, where precise control over lipid composition and particle size is critical for vaccine efficacy. Recent advances include the development of 3D-printed microfluidic devices and continuous flow systems for scalable production.

2.5.2 3D Printing of Nanoparticles

Three-dimensional printing technologies have been adapted for nanoparticle fabrication, enabling the creation of complex nanostructures with precise spatial control. Techniques such as two-photon polymerization and direct laser writing can produce nanoparticles and nanostructures with resolutions below 100 nm. 3D printing allows for the fabrication of personalized nanoparticle-based drug delivery systems with tailored release profiles and geometries optimized for specific administration routes.

2.5.3 AI-Driven Nanoparticle Design

Artificial intelligence and machine learning algorithms are being increasingly applied to predict and optimize nanoparticle properties. Machine learning models trained on large datasets of nanoparticle formulations can predict the optimal synthesis parameters for desired particle characteristics, significantly reducing the time and cost of formulation development. AI-driven design is particularly promising for lipid nanoparticles, where the vast chemical space of lipid combinations makes experimental screening impractical.^[22,23]

3. CHARACTERIZATION OF NANOPARTICLES

Characterization is a crucial step in the development of nanoparticle-based drug delivery systems because the physicochemical properties of nanoparticles directly influence their biological performance, therapeutic efficacy, stability, and safety. Comprehensive characterization helps in understanding particle size, morphology, surface charge, crystallinity, chemical composition, thermal behavior, drug loading capacity, and release characteristics. Regulatory agencies also require detailed characterization data to ensure product quality, reproducibility, and clinical safety.

3.1 Particle Size Analysis

Particle size is one of the most important parameters influencing nanoparticle behavior. It affects cellular uptake, biodistribution, circulation time, drug release kinetics, and targeting efficiency. Nanoparticles with smaller particle sizes generally exhibit improved cellular internalization and enhanced permeability through biological barriers.

Particle size distribution is commonly expressed in terms of mean particle diameter and polydispersity index (PDI). A narrow size distribution is desirable because it ensures formulation uniformity and reproducibility.

1. Dynamic Light Scattering (DLS)

Dynamic Light Scattering is the most widely used technique for measuring nanoparticle size in suspension. The method is based on the analysis of fluctuations in scattered light caused by the Brownian motion of particles. The diffusion coefficient obtained from these fluctuations is used to calculate the hydrodynamic diameter of nanoparticles.

Advantages:

• Rapid and non-destructive analysis
• Suitable for colloidal systems
• Provides particle size and PDI simultaneously.^[24]

3.2 Polydispersity Index (PDI)

PDI is a measure of particle size distribution uniformity. It indicates the degree of heterogeneity within a nanoparticle formulation.Lower PDI values indicate better formulation quality and stability.^[25]

Table no.2: Polydispersity Index (PDI)

3.3 Zeta Potential Analysis

Zeta potential is a measure of the electrical charge present on the nanoparticle surface. It is an important indicator of colloidal stability because surface charges generate electrostatic repulsion between particles, preventing aggregation.The zeta potential also influences interactions with biological membranes, protein adsorption, cellular uptake, and biodistribution.^[26]

Table no.3: Polydispersity Index (PDI)

3.4 Morphological Characterization

Morphological analysis provides information regarding nanoparticle shape, surface texture, aggregation state, and structural organization. Morphology significantly affects drug loading, release behavior, and biological interactions.

1. Scanning Electron Microscopy (SEM)

SEM uses a focused electron beam to scan the sample surface and generate high-resolution images.

Applications:

• Surface morphology analysis
• Shape determination
• Aggregation assessment
• Particle size estimation

Advantages:

• High-resolution imaging
• Large depth of field
• Excellent surface visualization

2. Transmission Electron Microscopy (TEM)

TEM involves transmission of electrons through a thin sample to obtain highly detailed images.

Applications:

• Accurate particle size determination
• Internal structure analysis
• Core-shell nanoparticle characterization
• Crystallinity assessment

Advantages:

• Extremely high resolution
• Direct visualization of nanoparticles
• Internal structural analysis

3. Atomic Force Microscopy (AFM)

AFM employs a nanoscale probe that scans the surface of nanoparticles to generate three-dimensional topographical images.

Applications:

• Surface roughness analysis
• Morphological characterization
• Mechanical property evaluation

Advantages:

• Three-dimensional imaging
• Minimal sample preparation
• High spatial resolution.^[27]

3.5 Structural Characterization

Structural characterization determines crystalline structure, phase composition, and molecular organization of nanoparticles.

• X-Ray Diffraction (XRD)

XRD is used to identify crystalline phases and evaluate crystal structure. The technique measures diffraction patterns generated when X-rays interact with crystalline materials.The Debye-Scherrer equation is commonly used to estimate crystallite size from XRD peak broadening.

Applications:

• Determination of crystallinity
• Phase identification
• Crystal size estimation
• Evaluation of drug encapsulation effects

Advantages:

• Non-destructive analysis
• High accuracy
• Quantitative assessment of crystallinity.^[27]

3.6 Chemical Characterization

Chemical characterization identifies functional groups, molecular interactions, and surface chemistry.

1. 1. Fourier Transform Infrared Spectroscopy (FTIR)

FTIR detects absorption of infrared radiation by molecular bonds, generating characteristic spectra for specific functional groups.

Applications:

• Drug-polymer compatibility studies
• Surface functionalization analysis
• Confirmation of nanoparticle formation
• Identification of chemical interactions

Advantages:

• Rapid analysis
• Minimal sample preparation
• High sensitivity
  2. Raman Spectroscopy

Raman spectroscopy provides molecular information based on inelastic scattering of light.

Applications:

• Molecular fingerprinting
• Structural characterization
• Surface chemistry analysis
• Detection of molecular interactions

Advantages:

• Non-destructive technique
• High specificity
• Suitable for aqueous samples.^[28]

3.7 Surface Area Analysis

Surface area is a critical parameter influencing drug loading, dissolution rate, and adsorption capacity.

• Brunauer-Emmett-Teller (BET) Analysis

BET analysis determines specific surface area by measuring gas adsorption onto the nanoparticle surface. Higher surface area generally results in improved drug loading capacity and enhanced dissolution.

 Applications:

• Surface area determination
• Porosity assessment
• Evaluation of mesoporous nanoparticles.^[29]

4. PHARMACEUTICAL APPLICATIONS OF NANOPARTICLES

Nanoparticles have revolutionized modern pharmaceutical sciences by providing innovative solutions to challenges associated with conventional drug delivery systems. Their unique physicochemical properties, including small particle size, large surface area-to-volume ratio, tunable surface characteristics, and ability to encapsulate a wide range of therapeutic agents, make them highly effective carriers for pharmaceutical applications. Nanoparticles can improve drug solubility, enhance bioavailability, prolong circulation time, facilitate controlled release, and enable site-specific drug delivery. As a result, nanoparticle-based systems have found widespread applications in drug delivery, cancer therapy, gene therapy, vaccine development, antimicrobial treatment, diagnostic imaging, and theranostics.^[30]

4.1 Targeted Drug Delivery

Targeted drug delivery is a major application of nanoparticles that improves therapeutic efficacy while reducing systemic toxicity. Drug-loaded nanoparticles circulate in the bloodstream and accumulate at diseased tissues or tumors through passive or active targeting mechanisms. After binding to target cells, they are internalized by endocytosis and release the drug in a controlled manner, resulting in enhanced treatment outcomes and reduced damage to healthy tissues.

1. 1. Passive Targeting

Passive targeting relies on the Enhanced Permeability and Retention (EPR) effect observed in tumor tissues and inflamed sites. Tumors possess abnormal blood vessels with large fenestrations and poor lymphatic drainage, allowing nanoparticles to preferentially accumulate within tumor tissues.

Advantages of passive targeting include:

• Increased drug concentration at the disease site.
• Reduced exposure of healthy tissues.
• Improved therapeutic index.
  2. Active Targeting

Active targeting involves surface modification of nanoparticles with ligands such as antibodies, peptides, folic acid, aptamers, or transferrin. These ligands specifically recognize receptors overexpressed on target cells, facilitating receptor-mediated uptake.

Examples include:

• Folate receptor-targeted nanoparticles for ovarian cancer.
• HER2-targeted nanoparticles for breast cancer.
• Transferrin-modified nanoparticles for brain targeting.^[31]

Figure 3. Schematic of Nanoparticle-Mediated Targeted Drug Delivery.