Advanced Methods for Nanoparticle Encapsulation: Novel Drug Delivery Systems (NDDS) for Enhanced Therapeutic Efficacy

1.1. Contextualizing Nanomedicine within Modern Therapeutics

Nanomedicine is a groundbreaking shift in the way modern medicine works by replacing nonspecific systemic delivery with targeted, local intervention using size-appropriate nanoparticles (NCs).8 This strategy forms the basis of Novel Drug Delivery Systems (NDDS), which are widely utilized for numerous difficult to treat diseases including cancer, cardiovascular disease, Alzheimer's disease, diabetes and HIV.9 The main purpose of NDDS development is to promote efficient drug loading for optimal therapeutic agent accumulation at a target site of disease with minimized systemic exposure and toxicity.8 The encapsulation process of any nanomedicine formulation is a key determinant to its therapeutic potential. Good encapsulation, including prevention of premature drug release and protection of sensitive payloads (e.g., nucleic acid and protein) from degradation in hostile physiological environments, is critical.1 The extent to which encapsulation occurs is the most important factor for therapeutic success. Indeed, the prevention of drug escape positively impacts the toxicity profile of DDS since it reduces systemic exposure to the free drug thus avoiding which may avoid on-target off-tumour toxicities. With strong encapsulation, the high buildup at the target is increased, which increases selective effectiveness.8 Therefore, beyond a manufacturing perspective itself, the selection of encapsulation method becomes one of the major factors for overall nanodrug safety and efficacy.

1.2. High Fidelity Encapsulation is a Must for NDDS

Nano capsules, which are submicron drug delivery systems consisting of a core containing drug(s) surrounded by polymeric shells, are designed to offer improved pharmacological benefits compared with free drugs.10 These advantages include prolonged drug delivery kinetics, increased selectivity and efficiency of the drugs, improved bioavailability, and successful drug de-toxification.10 However, optimal nanocarrier design is a balancing act of opposing necessities: the carrier shell must be kinetically stable enough to provide long-term systemic residence and prevent immune system recognition, yet also afford thermodynamic instability such that cargo release at the target pathology is both rapid and efficient.2 This requirement for engineered failure underscores an important design problem. On the contrary, therapeutic dose cannot be released in over-stabilized system and carrier clearance may chronically occur. Alternatively, defect of the integrity of the barrier leads to early leakage of active components.1 The challenge is to achieve environmental sensitivity of the carrier surface character in order for the therapeutic agent to be released if and only if prompted by cues characteristic of a pathological microenvironment, such as pH or redox potential changes.9 This finely-tuned instability point is useful in maximizing the therapeutic effect of ABPs while minimizing their toxicity as they circulate in blood.

1.3. Core Nanocarriers’, Classification: Structure and Applications

Drug delivery systems employing nanomaterials have been conventionally categorized into two broad groups according to their composition, i.e., organic/polymer-based and inorganic/metal-based.9

1.3.1. Organic and Polymer-Based Nanocarriers

As a general application in NDDS, organic and polymeric systems are commonly used owing to their biological compatibility, high drug loading, ability along with the controllable surface morphology & chemical composition. 9 Notable examples are polymer functionalized micelles, liposomes, vesicles and dendritic systems.9–11 Among them, polymers such as poly (lactic acid) (PLA) and poly(lactide-co-glycoside) (PLGA)-based devices are the most common types and they are also widely accepted by regulatory bodies (e.g., USFDA) due to their biocompatible nature and controlling ability of drug release for a long time period which is very effective in treatment of chronic diseases like cancer.9

1.3.2. Inorganic and Metallic Nanocarriers

Inorganic nanomaterials have found widespread applications because of their unique physical properties and functional potential. Systems in this class include Carbon Nanotubes (CNTs), Gold Nanoparticles (AuNPs), Quantum Dots (QDs) and Magnetic Nanoparticles (MNPs). 9 In particular, magnetic nanoparticles (MNPs) are very useful in that they can respond to an external magnetic field, and show selective accumulation with high efficiency and simultaneous imaging ability (theragnostic).13 Clinical approvals of these nanocarriers have been obtained in the treatment of numerous fatal diseases.9 The basic features and functions of these core nanocarriers are listed in Table 1.

Table 1: Classification and functional characteristics of nanocarriers.

2. Physical and Chemical Processes for Nanoparticle Synthesis

The choice of optimal manufacturing process for nanocarrier directly affect the physicochemical properties studies, such as morphological characteristics, size distribution and drug loading efficiency of such nanocarrier. Contemporary techniques focus on accessibility, control and efficiency.

2.1. Conventional Phase Separation and Precipitation Techniques

2.1.1. Nanoprecipitation (Solvent Displacement)

Nanoprecipitation, also known as solvent displacement, is been considered to be of easy and effective way for preparing polymeric nanoparticles. 14 In this process a solution containing the polymer and drug (dissolved in water miscible organic solvent) is mixed with an aqueous non-solvent in “one-pot” controlled manner. 14 The particle formation process begins with the fast diffusion of organic solvent, into non-solvent phase which results in polymer and drug to achieve oversaturation state and precipitation thereafter. It enables good controllability of the resulting nanostructure morphology, including complicated structures like core-shell and stacked lamellar or porous cores. 14 These properties can be controlled by tuning parameters like the starting materials (block copolymers vs. homopolymers) and rate at which the system will come to equilibrium: go through the glass transition, or crystallize. 14 Nanoprecipitation compares favourably to conventional emulsification methods that can involve energy-intensive techniques with the application of high-shear forces (for example, ultrasonication), or the use of un-desirable stabilizing surfactants. 14 Due to the relatively mild reaction conditions and its simplicity, this is one of the most popular methods to control polymeric nanostructures.

2.2. Interfacial Deposition Techniques

Interface deposition methods have aimed to generate the shell structure around the active drug particles in a self-organized manner mostly led by macromolecular interactions.

2.2.1. Complex Coacervation: Liquid-Liquid Phase Separation (LLPS) Principles

For example, coacervation is a liquid-liquid phase separation (LLPS) phenomena where solution of polymeric colloids undergoes spontaneous separation into two phases: polymer rich coacervate and a polymer diluted phase.3 The complex coacervation in particular is induced by the strong hydrophobic interactions of polyelectrolytes (polyanion and polycation) with opposite charges that eventually leads to charge neutralization and aggregation.3 This was proposed as a pH-dependent equilibrium, which is affected by the ionic strength and temperature.3 Compared to the all-synthetic polymer system, the use of biomolecules (proteins, nucleic acids and polysaccharides) for coacervation also contributes better biocompatibility and possibility to respond more effectively to physiological stimuli.3 This methodology offers a high encapsulation efficiency and great stability for sensitive or hydrophilic payloads, allows to sustain the release profile. 3 Because coacervation is based on gentle, aqueous-phase interactions with none of the high heat or toxic organic solvent volumes in much of chemistry.16 The natural biocompatibility and non-toxic processing of complex coacervation makes it a good candidate for the regulatory Quality by Design (QbD) guidelines, as it reduces solvent residual toxicity in addition to reducing hazards with product purity.3

2.2.2. Layer-by-Layer Assembly by Electrostatic and Hydrogen Bonding

The Layer-by-Layer (LbL) assembly is a powerful technique to fabricate well-structured encapsulation shells by the stepwise counterion adsorption onto a substrate which can be as simple as just one single nanoparticle and also an entire cell.4 The deposition is driven by several intermolecular interactions, mainly electrostatic attractions between polyanions and polycations, but also including hydrogen bonding and other non-covalent contacts.4 LbL provides excellent control over the composition, morphology, and thickness of the multilayered shell, which makes it possible to build complex multi-functional barriers. 17 This tunable control enables the design of chemically complex shells, and it places LbL as a reference platform to develop high-level layers that can incorporate multiple functionalities (protection, targeting, stimuli responsiveness) in a unique nano system. The final release kinetics of the drug payload can be strongly influenced by way of adjusting the composition of individual layers. 4 In addition, the resultant structure is possible to be tailored by materials or linkers that are sensitive to pathological environment (e.g., redox potential, pH, or biologics) for controlled and triggering payload release.4 This spatial manipulation of the chemical moiety enables the engineer to fortify a stable inner barrier for circulation protection, in conjunction with an environment-triggered outer layer for responsive release at the site of action, thus fulfilling the stability-release paradox in NDDS design.2

3. Next-Generation Encapsulation: Synthesis of Accurate and Bio mimicker Objects

3.1. Microfluidics: Nanoscale Monodispersity And Reproducibility. Transfer of DNA mediated by electrically charged vesicles Nature

Microfluidic devices are a major step towards the precision high power synthesis of nanomedicines, exploiting fluid dynamics in microchannels to enable fine grained, highly reproducible control over every stage in a synthesis process from nucleation through growth kinetics to emulsification.18 Such control is vital for the regulation of important quality attributes, such as NP size, surface characteristics and drug encapsulation efficiency.18 Microfluidics, being continuous flow-based systems, immediately overcomes one of the most significant hurdles in translational nanomedicine—batch-to-batch variability. 18 Improving the reliability and uniformity of nanoparticle production, microfluidics stands as the foundation for an efficient and scalable synthesis that offers high regulatory compliance in respect to reproducibility. 18 Advanced systems, such as acoustic microfluidic devices allow the synthesis of complex hybrid systems. For instance, the encapsulation efficiency surpasses 70% for coating nucleic acid-loaded poly (methyl methacrylate) (PMMA) NPs with their natural vesicles such as liposomes and Extracellular Vesicle (EVs), You et al., demonstrating its capability to finely control synthesis parameters and material feature toward custom-made delivery systems.20 The use of continuous flow processing is consequently one of the strategic choices to be implemented for developers that are looking for an efficient and scalable production.

3.2. Cell Membrane-Coated Nanoparticles (MBNPs)

Biomimetic strategy inspired creation of the Membrane-wrapped Biomimetic Nanoparticles (MBNPs), using natural cell membranes obtained from the host cells, as Red Cell Membranes (RCMs), Cancer Cell Membranes (CCMs) or Exosome membranes (EMs).5 This strategy overcomes the main obstacle of immune eliminatory, i.e., RES avoidance. The low immunogenicity comes with the natural membrane coating on carrier, which also greatly promotes the circulation time of carriers.5 MBNPs solve the so-called “PEG dilemma” of traditional drug delivery. Although such conventional PEGylated systems can prolong the circulating life time of vectors in blood, their cellular uptakes and transfection efficiencies are largely reduced. 5 MBNPs escape this trade-off by expressing nonendogenous don’t recognize and signals on their surface resulting in long-systemic circulation, while achieving increased-targeted uptake. 5 This action guarantees that drugs or genes are precisely delivered to target tissue without untimely release, degradation, or clearance, which means extensive biosafety and higher efficacy in vivo..5 This strategic choice in source of the membrane (e.g., RCMs for stealth, CCMs for homotypic targeting) enables a level of control over biodistribution that is unattainable by purely synthetic optimization of nanocarriers and novel among carriers constructed to strategically mimic native biological interfaces. 5 As surface properties are known to control stability as well as drug leakage 1, a cell membrane coating can produce the most biologically relevant barrier, thereby resulting in maximum fidelity encapsulation.

4. Modulation of drug release: kinetics and stimuli responsive properties

4.1. Performance Parameters: The DLC and EE

The potential healthcare utility of any NDDS is measured in terms of its cosmeceutical viability as assessed by the Drug Loading Content (DLC) and Encapsulation Efficiency (EE). High EE and DLC are important not only for financial aspect but actually for safety. Another drawback of such low DLC is that it requires administration of relatively high mass materials for the carriers (even in excess of 90% by weight) to achieve a deposit effect 10 which provides unnecessary exposure to parenteral excipients and risks for toxicity.10 Thus, EE and DLC are both essential factors in NDDS design.

4.2. Drug Release Kinetics and Modelling Pollutants Control: Drug Transport and Kinetics 291

The ultimate aim of regulating drug release profile is to maintain the concentration of drug at the patient’s bloodstream or target tissue at therapeutic window, that is, between minimum effective concentration (MEC) and minimal toxic concentration (MTC).2 The engineering goal for sustained release is to achieve a zero-order (independent of time) drug release profile in which the rate of drug released remains constant over an extended period.2 Based on drug release mechanism, a release system can be categorized into the four types: Diffusion-Controlled, Solvent-Controlled (swelling and osmosis), Chemical Reaction-Controlled (cleavage or degradation of carrier linkages), and Stimuli-Controlled.2 In diffusion-controlled release, which is the most observed case in matrix type nanospheres, drug molecules that are distributed within the polymer matrix diffuse out. 2 Such a mechanism is frequently accompanied by an initial burst release, followed by a decline in the rate of diffusion as distance increases. Control over the initial burst is very important because, often necessitating specific encapsulation as LbL-surface modification.4

4.3. Design of stimuli-responsive release systems (srNPs)

In association to pain and inflammation, the passive diffusion of drug molecules toward the target site suffers from some obstacles, such as inefficient reaction times, otologist intervention in the case of chronic treatments, enzymatic destruction or slow interaction with the receptors. To bypass these limitations intrinsic to passive diffusion and thus achieve a timely presence on the site of action for an efficient drug activity, Stimuli-Responsive Nanoparticles (srNPs) are designed to undergo a rapid modification in their morphology or integrity upon triggering by relevant cues.9

4.3.1. Internal (Pathological) Triggers

These are designed to target molecular markers specific to the site of disease. In oncology this often means using unique properties of the tumour microenvironment:

pH and redox potential:

Tumour tissue and intracellular endosomes/lysosomes frequently present a lower pH, which is acidic in nature, in comparison to normal tissues. A high intracellular reducing potential induced by an elevated concentration of glutathione is closely related.9 Acid-labile or redox-cleavable linkers that are employed in nanocarriers are programmed for endosomal and cytosolic disruption only, when exposed to these specific intracellular environments.

Cleavage by Enzymes:

Polymeric components bound through a peptide sequence that can be cleaved selectively by enzymes overexpressed by tumour cells or stromal tissue4 lead to highly localized drug elution.

4.3.2. External Triggers

Extrinsic stimuli are advantageous to allow remote, precise dictate of drug release on timing and location:

Magnetic fields:

This category is dominated by magnetic nanoparticles (MNPs). External magnetic fields enable the guidance and collection of them at a target location.13 Moreover, by the use of an alternating magnetic field energy is able to generate locally in this way (magnetic hyperthermia), and this might cause degradation or phase transition of temperature sensitive polymeric shells inducing drug release.13 This responsiveness is important for therapeutic gene delivery that requires selective localization and release.13

Temperature and Light:

Other external stimuli, such as a focused ultrasound or near-infrared light (NIR) can be used to produce local heating (photothermal effects), therefore leading to the on-demand release of incorporated agents from thermo-responsive carriers.9 The coexistence of passive targeting (such as the EPR effect) or active targeting (e.g., magnetic guidance) with site-specific, stimuli-triggered release is the most advanced application in NDDS.13 This combination strategy effectively increases the therapeutic concentration ratios of disease versus healthy between location of drug action, where we can thus minimize collateral damage and maximize selective efficacy so that the overall effective dose is lower.13

5. Translational science: Strategy in manufacturing and regulation

5.1. Scale-Up Limitations of Batch Processing Overcome

Moreover, the translation of nanomedicines from lab scale product to clinical use is frequently hindered by restrictions associated with conventional manufacturing methods. The conventional methods for micro-scale micronization and the coating, like spray drying and high temperature evaporation, usually produce particles that are too large, poorly controlled by fine structure/shape morphologies, incompletely removing solvents.16 The latter shortcomings as well as the relatively high cost and non-continuous operability are major obstacles for industrial practice and mass production.16

5.2. Supercritical Technology for Industrial Nanoencapsulation

An alternative to the conventional nanocarrier production for industrial purposes, supercritical fluid (SCF) technologies, and more specifically those based on supercritical carbon dioxide have developed into an efficient alternative.6 These are due to the fact that it makes use of distinctive controllable properties of (low viscosity, high diffusivity, variable solvent power) allowing an accurate control of critical parameters such as particle size, morphology and drug loading by acting only on operating conditions (pressure, temperature and flow rate).6

5.2.1. Rapid Expansion of Supercritical Solutions (RESS)

RESS uses as the solvent for the medicament. Particular are formed due to violent expansion of the solution on passing through a nozzle into a low-pressure chamber.6 This sudden drop in pressure results in an immediate lowering of solvent power, which generates high supersaturation and nucleation of fine particles followed by their precipitation. Although rests are great for micronization, it can only encapsulate soluble substances, and hence in general isn't suitable for complex biopolymers.6

5.2.2. Supercritical Antisolvent (SAS)

In the SAS process, acts as an antisolvent.6 The drug– and/or coating material are dissolved in the organic solvent that is then sprayed into the cell.6 The solvent power is dramatically decreased by the rapid penetration of into the organic solution, and consequently microparticles or nanoparticles are formed during precipitation.6 SAS has been demonstrated to be micronize APIs, in particular hydrophobic drugs and is associated with monolithic matrix systems.6 However, there is a serious material science problem using amorphous polymers such as PLGA where glass transition depression and plasticization can occur in the presence of making it difficult to prepare discrete, free-flowing powder particles.6

5.2.3. Extraction of Emulsions with Supercritical Fluids (SFEE)

SFEE was designed to circumvent the plasticization and morphology collapse that hampers SAS for processing amorphous polymers.6 The approach relies on preparing a primary emulsion (Oil-in-Water (O/W) or Water-in-Oil-in-Water (W/O/W) for hydrophilic drugs) in which the polymer and drug are both dissolved within an organic solvent.16 Supercritical text is then employed solely to extract the organic solvent of the emulsion droplets.16 Finally, after the solvent extraction material, which is also responsible for precipitating both the polymer and active substance in a controlled manner produces monodisperse micro- or Nano capsules dispersed in an aqueous phase.6 SFEE is especially appropriate for fragile substances like proteins and also made it possible to nano encapsulate some amorphous materials such as PLGA, PMMA, PCL etc.16 The fact that SFEE can reliably encapsulate these critical amorphous polymers without any morphological detriment (i.e., plasticization) serves as a crucial link to facilitate a high control, scalable transition of highly complex polymeric NDDS from the benchtop to industry scale manufacturing, satisfying key translational criteria.16 Therefore SFEE is a strategically needed process for an industrial feasibility.

Table 2: A Comparison of Advanced/Industrial Encapsulation Techniques

6. Regulatory Landscape and Quality Assurance

6.1. Classification and Difficulty of General Breathing Models

The translation of nanomedicines from the lab to the clinic is very well-regulated by regulatory agencies like FDA and EMA.7 The specific physicochemical attributes, as well as crossbreed character of most nano medicinal products, make both determining and taxonomizing nanoparticle-based therapies particularly daunting in some instances where nanomedicines themselves can function as a therapeutic agent with the properties of drugs or biologics or medical devices.19 Such a fuzzy regulatory state potentiates the approval pathway and emphasizes early and ongoing interaction with agencies.19 In addition, due to the dynamic nature of nanoparticles themselves, slight changes in process conditions result in significant differences in key characteristics of NPs including size, surface property and in vivo body distribution.19 This variability adds significantly to the perceived risk and demands rigorous analytical methods with well-defined limits that ensure product reproducibility and safety.19 Current regulations frequently fail to meet the prerequisites of nanomedicines and are associated with high regulatory ambiguity.19

6.2. Quality by Design (QbD) Approach in Nanomedicine

Quality by Design (QbD) is becoming a prevailing practice to control complexity and risks during nanomedicine development, especially for the purpose of marketing approval for such products.22 QbD requires quality to be designed into the product since its early development stages, which also implies a thorough understanding of how material properties and process parameters contribute to produce the final product.7 Under the QbD paradigm this implies the definition of rugged, well-characterized methods to be applied across the entire product lifecycle during a routine assessment for Critical Quality Attributes (CQAs).7 The introduction of advanced encapsulation techniques, including microfluidics and tunable SCF processes, are essential for a successful QbD approach. These technologies provide the process control and consistency required to accurately define and maintain CQAs, which in turn, that will allow risks related to size of particles and biodistribution variability to be reduced.18 Thus, the high-precision manufacturing could be applied successfully as a strategic move directly contributing to clinical translational victory through meeting regulatory criteria, which insisted on predictable and robust production.

6.3. Novel Characterization of Critical Quality Attributes (CQAs)

Regimes that regulate heavily are much more concerned with quality and safety of products.7 The evaluation of product quality mandates extensive characterization of the physicochemical characteristics, quality control parameters, and manufacturing process.7 This requires that state-of-the art analytical tools such as dynamic light scattering (DLS), electron microscopy (TEM/SEM) and mass spectrometry are utilised to precisely characterize particle size distribution, shape, stability and integrity under conditions which simulate the biological environment.19 Product safety evaluation is another key issue and requires broad preclinical studies on pharmacokinetics, biocompatibility, immunogenicity, biodegradation or bioaccumulation of nanoparticles and nanotoxicology.7 A major challenge here is the knowledge of what actually happens to the nanocarrier biologically, including how and if they interacts with immune system and if gets cleared fast by RES or unwanted buildup in nontarget organs.7 A summary table of critical CQAs and how advanced encapsulation meets translational challenge is provided in the table below.