Hamdi Nsairat,1 Zainab Lafi,1 Belal O Al-Najjar,1 Ali Al-Samydai,1 Fadi G Saqallah,2 Mohamed El-Tanani,1,3 Ghaleb Ali Oriquat,4 Bailasan Mohammad Sa’bi,4 Abed Alqader Ibrahim,5 Anthony Lee Dellinger,5 Walhan Alshaer6

1Pharmacological and Diagnostic Research Center, Faculty of Pharmacy, Al-Ahliyya Amman University, Amman, 19328, Jordan; 2Faculty of Pharmacy, Al-Zaytoonah University of Jordan, Amman, Jordan; 3College of Pharmacy, Ras Al Khaimah Medical and Health Sciences University, Ras Al Khaimah, United Arab Emirates; 4Pharmacological and Diagnostic Research Center, Faculty of Allied Medical Sciences, Al-Ahliyya Amman University, Amman, 19328, Jordan; 5Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, University of North Carolina at Greensboro, Greensboro, NC, USA; 6Cell Therapy Center, The University of Jordan, Amman, 11942, Jordan

Correspondence: Anthony Lee Dellinger, Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, University of North Carolina at Greensboro, P.O. Box 26170, 1400 Spring Garden Street, Greensboro, NC, 27402-6170, USA, Tel +1-336-334-5000, Email [email protected]

Abstract: The development of effective drug delivery systems is a key focus in pharmaceutical research, aiming to enhance therapeutic efficacy while minimizing adverse effects. Self-assembled nanostructures present a promising solution due to their tunable properties, biocompatibility, and ability to encapsulate and deliver therapeutic agents to specific targets. This review examines recent advancements in drug-based self-assembled nanostructures for targeted delivery applications, including drug-drug conjugates, polymeric-based architectures, biomolecules, peptides, DNA, squalene conjugates and amphiphilic drugs. Various strategies for fabricating these nanostructures are discussed, with an emphasis on the design principles and mechanisms underlying their self-assembly and potential for targeted drug delivery to specific tissues or cells. Furthermore, the integration of targeting ligands, stimuli-responsive moieties and imaging agents into these nanostructures is explored for enhanced therapeutic outcomes and real-time monitoring. Challenges such as stability, scalability and regulatory hurdles in translating these nanostructures from bench to bedside are also addressed. Drug-based self-assembled nanostructures represent a promising platform for developing next-generation targeted drug delivery systems with improved therapeutic efficacy and reduced side effects.

Keywords: self-assembly, targeted delivery, nanostructures, amphiphilic drugs, nanoconjugates

Introduction

Humans have utilized medicinal concoctions for thousands of years to improve the quality and longevity of life. In recent decades, the use of molecular therapeutic agents has increased significantly, especially in cancer diagnosis and treatment.1,2 However, with the increasing time and expense required for new drug development, interest has started to wane. The focus is increasingly shifting from the synthesis and discovery of new chemical entities to the development of innovative formulations and delivery systems for existing therapeutics, with the goal of enhancing clinical outcomes.3,4 In the context of cancer treatment, where numerous physiological, extracellular and intracellular barriers protect the body’s cells, drug delivery vehicles must be specifically engineered to circumvent the challenges. Nanostructured delivery systems have emerged as a promising strategy for improving drug bioavailability and precision targeting.5 This targeted drug delivery approach represents a significant advancement, enhancing therapeutic efficacy while minimizing systemic side effects.6,7

Among the many strategies in drug delivery, self-assembling supramolecular nanostructures have garnered considerable interest due to their tunable pharmacokinetic profiles and specificity in drug targeting.7 Drug-based self-assembled nanostructures represent a promising avenue for targeted delivery by utilizing the self-assembly phenomenon. Self-assembly refers to the spontaneous organization of molecules into well-defined structures.8 In these systems, therapeutic agents, such as drugs or bioactive molecules, autonomously organize into nanostructures or supramolecular assemblies stabilized through non-covalent interactions, including hydrogen bonding, hydrophobic interactions, or electrostatic forces. The primary goal is to develop nano-sized carriers or vehicles that enhance drug delivery efficiency, stability and targeting precision. By exploiting the intrinsic properties of the drugs themselves (wherein the drug serves as both the payload and the carrier), these systems eliminate the need for additional nondrug excipients, making them a compelling and efficient approach to advanced drug delivery.9–11

Drugs can be classified into synthetic compounds, steroids, peptides, sugars, nucleic acids and proteins based on the chemical structure of the active pharmaceutical ingredient (API).12 Numerous natural compounds and drugs exhibit the capability to self-assemble into nanostructures (Figure 1).12,13 For example, nucleic acids and peptides possess inherent structural properties and interactions that facilitate their self-assembly into nanostructures.14,15 This process is predominantly driven by noncovalent interactions, which enhance drug stability, enable controlled release and promote targeted delivery. Various types of drug self-assembly systems have been identified, including single-drug nano-assembled systems, drug-conjugate nano-assembled structures and multi-drug nano-assembled modules formed through the assembly of two or more drugs with additive conjugation.8

Figure 1 Schematic representation of self-assembled drug characteristics, formation and potential applications.

Given that the drug-based self-assembled nanostructures do not comprise traditional nanomaterials, these systems have promising potential for targeted drug delivery without the side effects associated with carrier materials.14 This approach simplifies the production process by reducing the need for quality control and monitoring related to nanomaterial’s type, quantity and potential interactions with the API and the body.16,17

Drug delivery strategies focus on passive and active targeting, both relying on the enhanced permeability and retention (EPR) effect to accumulate drugs in cancerous tissues.18 Tumor mass pores can allow for drug nanocarriers to penetrate and new targeted systems that use ligands like polyunsaturated fatty acids, folic acid, hyaluronic acid or oligopeptides for tumor recognition have been leveraged previously. However, notable challenges have included limited success for small ligands, enzymatic degradation in circulation and unclear tumor-targeting mechanisms.19 Active targeting involves attaching ligands to nanocarriers or drugs to bind specific biomarkers on tumor cells, improving drug localization, effectiveness and reducing side effects and blood concentration variation.20

An important consideration in the design and application of nanostructures is the formation of the protein corona. This dynamic biomolecular layer adsorbs onto the surface of nanostructures upon their introduction to a biological environment. In doing so, this interaction can have a profound impact on the pharmacokinetics, biodistribution, cellular uptake and immunogenicity of the nanostructure. Accordingly, the formation of a protein corona can alter the targeting efficiency of nanostructures by masking ligands or modifying surface characteristics, which can impact their ability to bind specifically to target receptors. Moreover, this layer can result in off-target effects by directing nanostructures to unintended tissues or via triggering immune responses in the patient. Future work aimed at addressing these challenges will require novel design approaches, such as engineering stealth surfaces with polyethylene glycol (PEG) or zwitterionic coatings to minimize protein adsorption. Furthermore, functionalizing nanostructures with targeting ligands or “pre-coating” them with specific biomolecules may help optimize these biological interactions. As research in this space continues to advance, understanding the intricacies of the protein corona will be essential for enhancing stability, functionality and safety of these nanostructures.

This review highlights the potential of drug-based self-assembled nanostructures in delivering safe and effective targeted therapies. Detailed examples and references are provided to highlight the versatility of these systems, their broad range of applications and the promising future directions for their development.

What are Self-Assembled Nanomaterials?

Self-assembly is a naturally occurring process where individual components autonomously organize into well-defined structures.14 This process is primarily driven by non-covalent interactions such as hydrogen bonding, hydrophobic interactions, electrostatic forces and π–π stacking.16,17,21,22 These interactions, commonly observed in natural phenomena such as the formation of cell membrane phospholipids, have been effectively utilized by researchers in the design of advanced drug delivery systems.15 Many drugs, especially those with amphiphilic or polymeric properties, exhibit the ability to self-assemble in aqueous solutions.13 Amphiphilic drugs, including hormones, proteins and surfactants, can form diverse structures in solution, determined by their inherent amphiphilic characteristics.5 By harnessing these self-assembly principles, researchers can develop sophisticated and efficient drug delivery vehicles for advancing the field of targeted therapeutics.

Harnessing Nature: The Science Behind Self-Assembled Nanostructures

In biological systems, numerous natural molecules demonstrate the ability to spontaneously form self-assembled nanostructures.23 For example, the self-assembly of lipids into liposomes facilitates efficient drug encapsulation and targeted delivery.24 This phenomenon is also observed in various biological processes, such as lipid particles forming oil droplets in water, polypeptides assembling into functional hemoglobin, the complex structures of ribosomal RNA and proteins, viral capsid formation and the organization of lipid bilayers within cellular membranes.15,25,26

Natural materials such as collagen, cellulose, protamine and silk have the ability to self-assemble into highly ordered nanostructures, providing diverse functionalities.22 A prominent example of self-assembly in physical chemistry is the organization of amphiphilic molecules into micelles, rods or liposomes. In this process, the hydrophobic tails and hydrophilic heads of lipids naturally arrange to form bilayers.14 Micelles are particularly apt at encapsulating hydrophobic drugs, while liposomes are capable of encapsulating both hydrophobic and hydrophilic drugs.27 Specifically, the lipid bilayer of liposomes incorporates hydrophobic molecules within the membrane, while the aqueous core encapsulates hydrophilic molecules. This dual functionality makes liposomes a versatile and promising system for delivering a broad spectrum of therapeutic agents.

Micelles are sphere-shaped amphiphilic nanoparticles (NPs) characterized by a hydrophobic core and a hydrophilic shell. In aqueous solutions, blocks of copolymers can assemble into fiber-like micelles;28 however, at low concentrations, these particles remain dispersed. As the concentration increases, these particles organize into a structured arrangement, a behavior known as the critical micelle concentration (CMC).29 Understanding and determining the CMC of amphiphilic molecules is essential for the formation of stable and functional micelles.30

Distinct molecules, like DNA and RNA, can serve as integral components in molecular self-assembling systems. These nucleic acids inherently self-assemble into diverse structures and complex aggregates, driven by multivalent interactions.31 This process often initiates in a less organized state, such as a solution, random coil or disordered aggregate, and gradually progresses toward a highly structured final state, such as a crystal or a folded macromolecule. This transition is largely driven by energy minimization and culminates in the formation of well-ordered and stable structures.32

The natural phenomena of molecular self-assembly enable the development of diverse nanostructures for drug delivery systems, including polymeric micelles, liposomes, nanocapsules and peptide-based formations. These engineered nanostructures can undergo further modification through the conjugation of specific functional groups, which enhances their stability, solubility and targeting capabilities.14,24,25

Classification and Formation: From Molecules to Nanostructures

Nanostructures can be categorized based on their structural characteristics and the nature of their self-assembly processes, which may be dynamic or static and involve the self-assembly of atoms, molecules or colloids.33–36 The classifications further extend to drug-based nanostructures, encompassing lipid-based, polymer-based and peptide-based assemblies, as well as classifications by size, shape and surface properties.12 Understanding these categorizations is essential for optimizing the performance and functionality of nanostructures in targeted drug delivery applications.37

Nanoparticles can be assembled from a variety of materials, including metals, polymers, proteins and lipids.5,22 Typically classified within a size range from 1 to 100 nanometers, the small dimensions of NPs provide a substantial increase in surface area, enhancing their functionality as drug delivery platforms. Drug-based self-assembled nanostructures offer a promising strategy for achieving targeted delivery.8,9 Their diminutive size confers multiple advantages, including improved drug bioavailability, higher concentration, enhanced solubility and greater stability. Additionally, NP-based drug encapsulation can elevate therapeutic efficacy by enabling targeted delivery to specific cell types and tissues, thereby minimizing off-target toxicity.6,38

For materials or molecules to assemble into nanostructures, non-covalent driving forces and interactions play a pivotal role in establishing stable and functional delivery systems.21,22 These forces facilitate both intramolecular and intermolecular self-assembly under defined conditions, such as solvation in aqueous environments. The chemical structure of drug molecules directly effects the nature of these interactions, influencing how drug molecules engage with each other and the surrounding water molecules. As shown in Figure 2, key interactions include hydrophobic and electrostatic interactions, hydrogen bonding and π–π stacking of aromatic groups.39 Together, these forces collectively determine the stability and functionality of the resulting nanostructures, enabling precise control over their assembly and behavior within biological systems.

Figure 2 Schematic representation of the prevailing forces that drive self-assemble.

Hydrophobic interactions are non-specific forces that are prevalent in biological systems. These interactions arise when molecules in aqueous environments rearrange themselves to bring hydrophobic regions into proximity, thereby minimizing contact with water and reducing the system’s overall free energy.40 Hydrophobic molecules naturally aggregate in aqueous solutions to achieve lower energy states.41 These interactions play a key role in the aggregation and self-assembly of NPs. In drug delivery, amphiphilic drugs or molecules that possess both hydrophobic and hydrophilic regions leverage these interactions to self-organize in aqueous environments, forming stable structures. This self-organization leads to the formation of micelles, liposomes and amphiphilic polymers, wherein the hydrophobic regions are sequestered within the core and shielded from the aqueous environment.8 This property represents an important determinant for the design of effective drug delivery systems.

Hydrophilic interactions are pivotal in stabilizing and functionalizing drug-based nanostructures. These interactions occur between the hydrophilic regions of drug molecules or their carriers, promoting the formation of a hydrated shell around the nanostructures, which is essential for their stability and solubility in aqueous environments.42 For example, in liposomes, the hydrophilic headgroups of lipid molecules orient themselves towards the aqueous surroundings, creating a stable aqueous core capable of encapsulating hydrophilic drugs. The presence of hydrophilic surfaces also helps maintain colloidal stability and prevents aggregation, which is often facilitated by the generation of a zeta potential around the nanostructure.43 A zeta potential exceeding ±30 mV is generally indicative of strong colloidal stability due to substantial electrostatic repulsions between particles. However, the threshold for stability depends on specific factors, including particle characteristics (eg, size, surface charge, and composition) and environmental conditions (eg, pH, ionic strength, and temperature). This electrostatic potential arises from charge distribution in the hydrophilic regions, creating a repulsive force that stabilizes the NPs in suspension. By harnessing hydrophilic interactions, researchers can design nanostructures that are more robust, stable, soluble and efficient, optimizing their use as delivery systems.

Electrostatic interactions between charged molecules represent another driving force that is crucial in the self-assembly process.44 These interactions occur when oppositely charged molecules attract each other, facilitating the formation of stable, self-assembled structures. For example, nanostructures can spontaneously organize through the electrostatic attraction between positively charged peptides or polymers and negatively charged nucleic acids.45 This fundamental principle is widely applied in the design of nanocarriers for drug delivery, where electrostatic interactions contribute to the stabilization and compaction of the therapeutic agents into functional delivery systems. These interactions play an important role in enhancing the structural integrity and overall performance of self-assembled nanostructures in biomedical applications.

Van der Waals forces are weak intermolecular forces that include both attractive and repulsive interactions between atoms, and molecules. Unlike covalent and ionic bonds, these forces are caused by the fluctuation in particle polarizations, which contribute to the stability of nanostructures.46 Despite their relatively weak nature, Van der Waals forces fulfill a critical function in the self-assembly of NPs and hydrophobic drugs with amphipathic nature.47 These forces facilitate the aggregation and stabilization of nanostructures, improving functionality and structural integrity.

Hydrogen bonding is a specific type of dipole–dipole interaction that occurs when a hydrogen atom is shared between electronegative atoms such as nitrogen, oxygen or fluorine. These bonds can form within a single molecule (intramolecular) or between different molecules (intermolecular), significantly influencing the physicochemical properties of polymeric compounds.48 For example, in the formation of double-stranded DNA, hydrogen bonds between adenine-thymine and guanine-cytosine base pairs ensure the stability and specificity of the helical structure. In proteins, hydrogen bonding is crucial for forming alpha-helices and beta-sheets, which are integral to their three-dimensional conformations. Hydrogen bonding is a key driving force for the self-assembly of various molecules, including polymers, peptides and nucleic acids.49,50 It stabilizes specific secondary structures by maintaining the spatial arrangement of these molecules, enabling the formation of functional and well-ordered assemblies.

Aromatic stacking, or π–π stacking, is a crucial interaction in the self-assembly of molecules containing aromatic rings. These interactions occur between the electron-rich π-systems of aromatic rings, leading to the formation of stable, stacked arrangements.51 Such stacking interactions are prevalent in molecules with multiple aromatic rings, such as DNA, where they contribute to the structural integrity of the double helix. Pi–pi (π–π) interactions are distinguished by their strong yet non-destructive nature, making them particularly effective in stabilizing self-assembled nanostructures. This stability is essential for enhancing the encapsulation efficiency and controlled release of therapeutic agents. For example, in nucleic acids and peptide-based conjugates, π–π stacking interactions between aromatic nitrogenous bases or aromatic amino acids serve a fundamental role that drives the formation, organization and structural integrity of self-assembled systems.52 These interactions are necessary for the design of drug delivery systems that rely on the precise and stable arrangement of molecules to function effectively within complex biological environments.

Certain polymers exhibit sensitivity to environmental changes, such as pH or temperature, which can trigger self-assemble processes. For example, pH-responsive polymers like poly(acrylic acid) (PAA) alter their conformation or charge state in response to pH fluctuations.53 This dynamic behavior allows these polymers to assemble or disassemble based on the prevailing pH conditions, making them versatile for tailored applications. Similarly, temperature-responsive polymers like poly(N-isopropylacrylamide) (PNIPAM) undergo a transition from a soluble to an insoluble state as temperature changes, initiating the process of self-assembly.54 These responsive properties enable precise control and modulation of polymer-based nanostructures, creating intelligent systems capable of specific responses under distinct environmental conditions. The adaptability of these responsive systems holds significant promise for various applications, particularly in drug delivery and the design of smart materials. By leveraging their environmental responsiveness, these polymers enable controlled drug release and improved targeting, laying the foundation for advancements in therapeutic and material science innovations.

Fabricating Nanostructures: Preparation Techniques for Precision Drug Delivery

Drugs can self-assemble through various mechanisms, requiring specific steps and conditions to ensure the successful formation of stable and functional nanostructures.55 Numerous methods, such as solvent evaporation, emulsification and thin-film hydration, can be used to prepare self-assembled nanostructures,56 thin-film hydration.57 Each technique offers control over critical parameters such as size, shape and drug loading capacity, which are critical for achieving targeted drug delivery.58,59 Thin-film hydration methods are suitable when the drug conjugate possesses either hydrophobic or amphipathic moieties, allowing for their dissolution in a suitable organic solvent.60 Next, the organic solvent is gently evaporated under reduced pressure to create a thin film layer. The obtained thin film is subsequently hydrated with an aqueous buffer solution to generate the nano-assembly. Reverse-phase evaporation methods are typically used as an alternative, forming a water-in-oil nano emulsion, whereas the injection methods involve dissolving the hydrophobic active agents with suitable solvent and then rapidly injected into an aqueous phase.61 The preparation process can generally be divided into four key steps:

(1) Selection of Materials: Identify appropriate drugs or materials capable of forming nanostructures, ensuring compatibility with self-assembly requirements and targeted applications.7

(2) Design and Formation: Develop the structural design and execute the formation of self-assembled particles under controlled conditions to achieve the desired nanostructures.

(3) Characterization and Optimization: Conduct physical and chemical characterization to assess the properties of the nanostructures and optimize their performance. Fundamental characterization techniques include dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM) and spectroscopy, which analyze particle size, morphology, stability, drug loading and release profiles.62 Surface modifications, such as covalent conjugation, electrostatic interactions or physical adsorption may be applied to improve targeting specificity, enhance stability or control drug release efficiency.63

(4) Biological Activity Assessment: Evaluate biological activity in both in vitro and in vivo systems to assess biocompatibility, efficiency, drug release, cellular uptake and targeting ability.64 In vitro studies may include release kinetics, cell viability assays and cellular uptake experiments. In vivo assessments focus on biodistribution, pharmacokinetics and therapeutic efficacy using relevant animal models systems to confirm the functional and therapeutic potential of the nanostructures.65,66

Engineering Effective Nanostructures: Morphology and Stability

Drugs (APIs) can be categorized based on their chemical structure into synthetic compounds, steroids, peptides, sugars, nucleic acids and proteins.12 Numerous natural compounds and drugs exhibit the capacity for self-assembly into nanostructures.13 For instance, nucleic acids and peptides inherently possess structural and interactive properties that facilitate their self-assembly into nanostructures.14,15 This process, predominantly driven by noncovalent interactions, contributes to enhanced drug stability, controlled release and targeted delivery. Drug self-assembled constructs can take diverse forms, including single drug nano-assembly modules, drug conjugate nano-assembled modules and multiple drug nano-assemblies formed by the assembly of two or more drugs with additive conjugation.8

The structural morphology and integrity of self-assembled drug nanostructures are critical determinants of their performance.62 Morphology pertains to the shape, size and arrangement of nanostructures, which may include spherical micelles, vesicles, fibers or hydrogels. Structural integrity, on the other hand, relates to the stability of these drug-based nanostructures and their capacity to preserve their configuration during storage and when interacting with biological environments.30

Structural morphology relates to the study of particle forms and shapes, which are influenced by material types and intermolecular forces. The shape and uniformity of particles serve a significant role in determining their performance and interactions with biological systems.67 Nanostructures can be engineered in various three-dimensional forms, including spheres, rods, fibers, vesicles and micelles.68 The selection of a specific shape is guided by the nature of the carrier, drug and targeting requirements. For example, spherical nanostructures are favored for their high drug loading capacity, controlled release and the potential for passive targeting through the EPR effect. Fibrous or nanotubular structures, in contrast, enhance cellular uptake and mimic the extracellular matrix, making them suitable for tissue engineering applications.69 Morphology can be modified through precise control of self-assembly conditions, including the selection of specific solvents, concentration, temperature and the inclusion of additives or surfactants. Particle morphology can be characterized using techniques such as electron microscopy, DLS, and small-angle X-ray scattering.70

Nanostructures must maintain their structural integrity and functionality without degradation or damage to ensure stability, efficient encapsulation and precise targeting capabilities of the formulated NPs.71 Common stability challenges during storage, such as aggregation, disintegration, phase separation and drug leakage, can compromise shelf life. Additionally, maintaining stability under varying physiological conditions, such as pH changes, enzymatic degradation and immune system opsonisation, is critical.72,73 To enhance the stability of nanostructures, it is important to optimize formulation parameters, incorporate additives and apply surface modifications. Various techniques can be used to evaluate the integrity of self-assembled nanostructures, such as fluorescence and infrared spectroscopy, and differential scanning calorimetry. Stability testing under relevant physiological conditions can be conducted through in vitro and in vivo release studies.16,72

Drug-Based Self-Assembly: A Novel Approach in Nanomedicine

Bioavailability is a major factor that influences the efficacy of anticancer therapies. Advanced drug delivery technologies, such as drug nanocarriers, have been developed to enhance the therapeutic potential of encapsulated chemotherapeutic agents by mitigating undesirable features and improving pharmacokinetics and tissue distribution.74–78 Despite their promise, many nano-therapies face significant hurdles in clinical translation, including low encapsulation efficiencies, compromised stability and the requirement for high volumes of non-drug excipients during NP production.79,80 Additionally, NPs are often sequestered by the reticuloendothelial system (RES), leading to off-target accumulation in organs such as liver, lung and brain. This sequestration can result in inflammation driven by NP-induced oxidative stress.81 Furthermore, the safety and toxicity profiles of some nanomaterials remain insufficiently characterized, posing challenges for regulatory approval and clinical application.82

These limitations are critical to achieving the appropriate therapeutic window.83,84 To overcome these constraints, a novel approach utilizing drug-based nanostructures has been developed. This approach leverages the ability of medications to self-assemble into supramolecular structures, forming stable nanocomposites. As described in Classification and Formation: From Molecules to Nanostructures, the primary driving mechanisms in self-assembly include noncovalent interactions (ie, van der Waals forces, hydrophobic effects, electrostatic interactions, hydrogen bonding, π–π stacking interactions, coordination bonding, and solvation and hydration forces).77 This strategy enables the development of self-delivering nanomedicines characterized by high and consistent drug content.85 The emergence of new nanocarriers that allow alternative dosing routes and reduced toxicity marks a significant progress in cancer treatment options.86 This approach confers novelty, especially when encapsulation enables high drug loading content, enhances anticancer activity and facilities targeted localization of drugs within cancer cells. Notably, this strategy offers the potential to mitigate drug resistance by engaging multiple pathways while reducing systemic toxicity compared to free drugs.

Amphiphilic Drugs: Dual-Function Molecules for Targeted Delivery

Amphiphilic drugs exhibit a dual nature, possessing both hydrophobic and hydrophilic characteristics and have an inherent capacity to self-organize into well-defined nanostructures.40,87 Early research in the 1950’s on drug self-aggregation demonstrated that penicillin and streptomycin salts could form colloidal micelles in aqueous solutions, as evidenced by surface tension measurements.88 Subsequent studies in 1971 utilizing 1H NMR confirmed that hydrophobic interactions drive this self-aggregation.89 This phenomenon was extended by Attwood and Argawal to include synthetic penicillins (ie, flucloxacillin and cloxacillin), which exhibited micellar properties in both water and isotonic saline.83 In drug delivery, the amphiphilic properties of these compounds dictate their potential for solubilization, bioavailability, integration into lipid membranes, transport characteristics and release kinetics from formulations.40 Moreover, amphiphilic drugs can be customized to interact with plasma proteins such as albumin or lipoprotein, enhancing their functionality and therapeutic potential.90,91

A limited number of studies have elucidated the capability of amphiphilic drugs to self-assemble into NP structures. Most recently, a study by Efthymiou et al (2021) demonstrated self-assembling properties of the hydrochloride salts of adiphenine, pavatrine and amitriptyline in aqueous solutions. Confirmed using small-angle X-ray scattering at concentrations above the CMC, these drugs formed micelles with an oblate spheroidal shape. While all three drugs exhibited a closed aggregation pattern, their amphiphilic nature resulted in pH sensitivity, leading to an increased micelle charge at higher drug concentrations.40 Despite offering several advantages over conventional methods, amphiphilic self-assembling drug delivery systems face competition from established technologies such as liposomes and polymeric NPs, each possessing distinct strengths and limitations. Recent advancements in computational tools and biomimetic approaches present promising opportunities to enhance the precision and efficiency of these systems, enabling more tailored design and optimized performance.

Barbosa et al (2008) explored the self-assembly behavior of two phenothiazine drugs, chlorpromazine (CPZ) and trifluoperazine (TFP), in aqueous solution using small-angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR). SAXS analysis demonstrated that CPZ molecules self-assembled into an orthorhombic cell basis configuration, forming nano-crystallites with aggregation numbers ranging from 60 to 80. Simulations of the EPR spectra using 5- and 16-doxyl stearic acids attached to aggregates provided insights into dynamic and magnetic characteristics.92 In related studies, edelfosine and fulvestrant were shown to self-assemble into nanostructures via the nanoprecipitation method, achieving high encapsulation efficiencies of 80% and 84%, respectively, for two hydrophobic agents. The resulting nanostructures had average sizes of 224.3 ± 1.8 nm and 247.3 ± 3.3, with zeta potentials of −17.3 ± 1.06 and −23.1 ± 3.51 mV. These nanostructures showed enhanced cellular uptake and penetration, with improved anticancer activity and the ability to induce apoptosis, particularly in estrogen receptor positive (ER+) breast cancer cell lines.93

Drug–Drug Conjugates

Self-assembling drug–drug conjugates represent an innovative approach in cancer therapy, aimed at enhancing drug delivery and therapeutic efficacy while minimizing adverse side effects.94 By applying the principles of nanotechnology and molecular design, researchers have developed systems that self-assemble into NPs, nanofibers, and other nanostructures capable of targeting specific tissues and releasing therapeutic agents in a controlled manner.95 These drug conjugates offer additional advantages beyond spontaneously forming NPs in aqueous environments, simplifying the preparation process, concentrating drugs within target tissues and potentially reducing systemic toxicity.96 Moreover, NPs derived from these conjugates can be engineered to enhance the penetration of small molecules through physiological barriers, thereby enhancing their pharmacological performance.

The concept of drug–drug conjugates is pivotal in contemporary pharmaceutical research, highlighting their ability to self-assemble through non-covalent interactions into NPs. Alternatively, self-assembly can be achieved by chemically modifying the drug into a prodrug, incorporating a non-toxic hydrophobic cleavable moiety. These prodrugs then assemble into nanoparticulates. This dual self-assembly mechanism highlights the versatility and potential of drug–drug conjugates in advancing nanomedicine.97 Building on these principles, Zhou et al developed a self-sufficient bi-prodrug nanomedicine technique to create a minimalist drug nanoplatform designed to enhance immunotherapeutic efficacy in chemotherapy. Gemcitabine (GEM) and 1-methyl-tryptophan (1MT), recognized for their bioactivity, were synthesized into a bi-prodrug molecule (GEM-1MT). These GEM-1MT bi-prodrug molecules demonstrated a unique ability to self-assemble into waste-free NPs for cancer treatment. This self-assembling capability significantly enhanced the overall therapeutic efficacy of combined chemo-immunotherapy. The bi-prodrug nanomedicine strategy introduces a novel approach for the deliberate design of straightforward drug nanoplatforms that improve the therapeutic outcomes of both immunotherapy and chemotherapy.

Numerous self-assembly inducers that can conjugate with drugs, such as hyperbranched poly(ether-ester), polyethylene glycol (PEG), hyaluronic acid, heparin and squalene, have been extensively reviewed previously by Fumagalli et al (2016).96 Expanding on these insights, Zhou et al (2023) investigated the self-assembly of bis(3-(pyridin-2-yl) phenyl) palladium(II) dimers for use in photo-dynamic therapy (PDT) as an anticancer treatment.98 The resulting self-assembled nanorods were evaluated against 3-dimensional A549 and A375 multicellular spheroidal models, yielding an EC50 value of 0.20 µM under irradiation. In vivo studies in mice with A375 tumors revealed high liver accumulation of the nanoconjugates, with lower levels in the heart, kidneys and lungs. This biodistribution pattern suggests prolonged bloodstream retention, leading to greater accumulation in tumor cells.98 Similarly, a platinum-containing prodrug was examined for its anticancer activity. This dimer was synthesized using cisplatin and a short peptide designed as a substrate for the phosphatase-catalyzed dephosphorylation. Upon dephosphorylation, the prodrug self-assembled into a nanotube hydrogel via π–π stacking and hydrogen bonding, forming structures with a diameter of 10 nm. This nanoconjugate effectively delayed cancer cell regrowth in 4T1 xenografted mice while significantly reducing liver and kidney accumulation and toxicity compared to free cisplatin. Furthermore, all treated mice maintained stable body weights throughout the treatment phase.99

Wang et al linked paclitaxel (PTX) dimers using a glutamic acid linker (Glu-PTX2), achieving a high PTX content of 88.9 wt.%. The Glu-PTX2 conjugates showed an ability to self-assemble into NPs (Glu-PTX2 NPs) in aqueous solution, significantly increasing their water solubility. These Glu-PTX2 NPs were internalized by cancer cells, where they exerted potent cytotoxicity. This innovative platform suggests that Glu-PTX2 NPs could serve as a promising alternative to free PTX with improved solubility and therapeutic efficacy.100

Continuing this trajectory, the second mitochondria-derived activator of caspases (SMAC), a pro-apoptotic protein, was conjugated with doxorubicin to create a self-assembling nanoconjugate that was capable of in vivo cleavage by cathepsin B, an enzyme highly expressed in cancer cells. These self-assembled nanoconjugates were stabilized through π–π stacking and hydrophobic intermolecular forces, resulting in the formation of spherical nanoparticulates with an average size of 221.8 nm. The SMAC-doxorubicin nanoconjugate demonstrated potential to address drug resistance in chemotherapy by delivering both therapeutic moieties simultaneously to their target sites in vitro. Furthermore, the nanoconjugate showed a 2.74-fold higher accumulation in Balb/c mice breast tumor within six hours of treatment.101

Doxorubicin–doxorubicin conjugates have been shown to self-assemble through π–π interactions. These di-doxorubicin conjugates were synthesized using either a disulfide linker102,103 or ester linkage.97 The resulting self-assembled nanoconjugates displayed an average size ranging from 75 to 180 nm with encapsulation efficiencies between 60% and 80%. Disulfide-linked conjugates showed higher cellular uptake, but lower cytotoxic activity compared to doxorubicin liposomes and free doxorubicin against MCF-7 cell lines. Notably, the disulfide-linked conjugates exhibited superior efficacy in female nu/nu mice xenografted with MCF-7 tumors, showing greater specificity for tumor cells over normal healthy cells.102,103 The release of doxorubicin from ester-linked nanoconjugate was found to be pH-dependent, with higher release rates in acidic microenvironments, thereby enhancing the targeting of cancer cells while sparing normal healthy cells. Further in vitro assessment using A549, HepG2, and MCF-7 cells revealed half-maximal inhibitory concentration (IC50) values of 7.69, 8.62, and 10.78 μg/mL, respectively.97 Recent advancements in the chemical modification of certain drugs, summarized in Table 1, highlight the potential for improved therapeutic performance through structural modifications.97–99,101–109

Table 1 Overview of Drug–Drug Conjugates: Physical Characteristics, Applications and Key Findings

The future of drug–drug conjugates represents a promising avenue in cancer treatment and holds great potential for improving therapeutic outcomes and overcoming drug resistance. However, to fully realize their clinical potential, it is imperative to overcome key challenges in formulation design, manufacturing and clinical translation. Progress in the field will depend on coordinated and collaborative efforts between researchers, clinicians and industry stakeholders to advance the technology and deliver its benefits to cancer patients.

Phytochemicals-Based Self-Assembly: Natural Solutions for Advanced Nanomedicine

In recent years, there has been a growing emphasis on investigating the active components present in traditional herbal medicine.110 Throughout history, compounds derived from plants, animals, fungi and microorganisms have served as pivotal elements in clinical drug discovery, particularly in the development of anticancer and anti-infective agents.111 The application of nanotechnology has emerged as a promising avenue for delivering natural phytochemicals and precisely regulating drug release within the body.112 Natural phytochemicals with self-assembly capabilities, such as flavonoids, terpenes,113 alkaloids114 and anthraquinones,13 have been identified as promising candidates for nanostructure development.115 These compounds exhibit an intrinsic ability to spontaneously organize monomers or multimers into well-defined nanostructures through noncovalent interactions in aqueous environments.116 This supramolecular noncovalent interaction, driven by the principle of minimum energy and the attractive forces between molecules, highlights the unique self-organizing behavior of these molecules.117 As a result, using natural phytochemicals as building blocks for the design of self-assembled functional nanostructures has emerged as a focal point in recent research.118 Among the diverse classes of phytochemicals (Figure 3), this section examines the distinctive types essential for self-assembly.

Figure 3 2D chemical structure of different natural compounds examples with Self-Assembling Nanostructures properties (Prepared by ChemDraw®).

Amongst this array of phytochemicals, steroids and terpenoids possess unique structural diversity and pharmacological significance. Steroids, which are characterized by a cyclopentane-poly(hydrophenanthrene) nucleus, possess a rigid hydrophobic backbone, flexible alkyl side chains, and multiple chiral centres. Terpenoids, the most abundant natural phytochemicals, utilize isoprene as a basic structural unit, with tetracyclic and pentacyclic triterpenes being the most common.119 Despite being homologous, these compounds exemplify how specific molecular architectures can influence self-assembly behavior and therapeutic potential.

Andrographolide, an active compound derived from Andrographis paniculata, is a terpenoid known for its notable anti-inflammatory and anticancer properties. Structurally, it is classified as a diterpene lactone, featuring a cis-1,3-diol configuration with hydroxyl groups at the 5-hydroxymethyl and 6-hydroxyl positions. Recent advancements in nanotechnology have explored its potential for drug delivery applications. For instance, Kim et al synthesized nanostructures by forming borate bonds between cis-1,3-diol of andrographolide and hydrophilic polymerized phenylboronic acid (pPBA), demonstrating pH-responsive controlled-release systems.120 Another approach utilizing glycyrrhizic acid as a building block to develop andrographolide-based nanostructures showed significant enhancements in both solubility and anticancer effects.121

Ginsenoside Rb1, a bioactive compound derived from Panax ginseng, has demonstrated the ability to self-assemble into stable NPs alongside anticancer drugs. These NPs feature hydrophilic head regions that facilitate aqueous connectivity while effectively isolating hydrophobic substances within the core. The stability and integrity of these nanostructures are further reinforced by π–π stacking interactions, which serve to create a robust system that does not induce toxicity or adverse effects, making ginsenoside Rb1-based green NPs a promising platform for the delivery of insoluble drugs.122

Steroids, which are abundant in biological systems, have been extensively explored for their potential in nanostructure self-assembly, particularly tetracyclic steroids characterized by their distinctive 6-6-6-5 ring structure. Sterols like ergosterol, known for their anticancer activity and self-assembly capability, have been utilized to create nanodrugs in combination with chlorin e6 (Ce6). These Ergo-Ce6 NPs exhibit remarkable phytotoxicity, increased blood circulation, excellent biocompatibility, prolonged tumor retention, biodegradability and low toxicity, making them a promising platform for targeted cancer therapies.123

Alkaloids are naturally occurring nitrogen-containing chemicals found in plants, fungi, and certain marine creatures. Alkaloids have a variety of chemical configurations and are frequently lipophilic, allowing them to interact efficiently with biological systems.124 Recently, alkaloids have garnered interest in drug delivery due to their propensity to conjugate with nanocarriers, which can result in stability, solubility and bioavailability enhancements.125 The ability of alkaloids to create self-assembled nanostructures allows for a more focused and controlled distribution that can lead to improved therapeutic efficacy with less adverse effects.126

Continuing this discussion of structurally diverse phytochemicals, alkaloids such as berberine exhibit remarkable self-assembly capabilities that contribute to their therapeutics. Berberine, a key antibacterial component of Coptis chinensis Franch, has gained attention for its ability to form nanostructures, driven by its polyaromatic ring structure and quaternary ammonium ions.127 Inspired by traditional Chinese medicine combinations, researchers successfully synthesized berberine-cinnamic acid NPs, which demonstrated superior inhibition of multidrug resistance compared to both berberine alone and control groups.13,128

Paclitaxel, a diterpene alkaloid derived from Taxus chinensis, is widely used in the treatment of breast and prostate cancers. However, its clinical application has been hindered by challenges such as water insolubility and multidrug resistance.129,130 To address these limitations, Cheng and Ji developed paclitaxel-sulfur-sulfur-Berberine (PTX-ss-BBR) NPs, which effectively accumulate in the mitochondrial and exhibit potent anticancer activity by inducing cell cycle arrest in the G2/M phase.131 Additionally, the combination of water-soluble vitamin E succinate with insoluble paclitaxel has been shown to self-assemble into paclitaxel-ss-VitE NPs through disulfide bonding. These NPs demonstrate significant pharmacological properties, including efficient hydrophobic drug loading and enhanced therapeutic efficacy.132 Another alkaloid, camptothecin, which is a quinoline alkaloid, has been shown to have potent antitumor activity, however has seen limited clinical utility due to challenges associated with poor solubility and stability. To overcome these obstacles, researchers have focused on developing effective nanodrug delivery systems. One such approach involves the self-assembly of hydroxycamptothecin and doxorubicin into hydroxycamptothecin-doxorubicin NPs. These NPs exhibit a morphological transition from nanorods to spherical particles over time, a process influenced by the molar ratio of doxorubicin to hydroxycamptothecin.133

Building on the self-assembly capabilities exhibited by alkaloids, flavonoids represent another class of natural compounds with significant pharmacological and structural potential. Known for their roles in managing cancer, neurodegenerative disorders and inflammatory diseases, flavonoids are increasingly recognized for their ability to self-assemble into functional nanostructures.134 This property enables them to serve as versatile building blocks in nanotechnology, further broadening the scope of natural phytochemicals in drug delivery and therapeutic applications.125 Baicalin and wogonoside, prominent bioactive flavones derived from Scutellaria baicalensis, exhibit self-assembly capabilities when combined with berberine, resulting in the formation of two distinct nanostructures.135 Li et al reported that baicalin-berberine NPs exhibited superior antibacterial activity compared to wogonoside-berberine nanofibers (NFs), a distinction attributed to differences in the spatial configuration and the underlying driving forces that influenced self-assembly mechanisms.136 While hydrophobic and electrostatic interactions predominantly drive the formation of baicalin-berberine NPs, the self-assembly of wogonoside-berberine NFs relies exclusively on potent hydrophobic forces. Notably, baicalin-berberine NPs initially form a one-dimensional unit, which subsequently evolves into a three-dimensional structure through interactions between hydrophilic glucuronic acid and the hydrophobic parent nucleus.136

Quinone compounds represent yet another diverse group of natural phytochemicals with self-assembling capabilities that have shown significant potential in nanomedicine. Quinones, encompassing benzoquinones, anthraquinones, phenanthrenequinones and naphthoquinones, are well recognized for their antibacterial and anticancer properties.137 Their propensity to form nanostructures stems from the presence of polyaromatic rings and hydrogen bonding interactions. Doxorubicin, a representative quinone compound with a hydrophilic amino sugar and a hydrophobic anthraquinone group, can form dimers or oligomers that act as building blocks for self-assembled nanodrugs.133 Anthraquinone, exemplified by rhein, exhibits self-assembly via hydrophilic, π–π-stacking, and hydrophobic interactions. In alkaline solutions, rhein molecules polymerize into dimers and aggregates, forming nanofibers through electrostatic repulsion.138 Furthermore, rhein can self-assemble with berberine into NPs through hydrogen bonds and π–π-stacking, creating a layered framework.13 Moreover, rhein hydrogels offer the advantage of molecular modification-free slow-release drug functions, introducing innovative concepts for nanomedicine design.137 Hypocrellin, a quinonoid derivative, demonstrates self-assembly with human serum albumin into NPs through hydrophobic interactions.139

Polysaccharides, the most abundant natural phytochemicals, exhibit antitumor, antioxidant and various therapeutic properties, making them promising candidates for nanostructure construction due to their biocompatibility, biodegradability and functional groups.140,141 While polysaccharides themselves do not inherently self-assemble, their multiple-hydroxyl groups facilitate hydrogen bonds, promoting orderly molecular arrangement. Researchers have enhanced the self-assembly potential of polysaccharides by modifying hydrophilic sugar groups with hydrophobic aromatic or alkyl chains, resulting in amphiphilic molecules that promote self-assembly in solutions.142,143 For instance, inulin, extracted from chicory root, forms spherical NPs and has shown potential in spinal cord injury treatment and dual cancer therapy when combined with curcumin.144–146 Similarly, pectin, another important polysaccharide, functions as a colon-specific drug-delivery material, effectively transporting drugs like dihydroartemisinin and hydroxycamptothecin to tumor sites.147

Beyond the commonly employed natural phytochemicals, unique molecular structures are gaining attention for their potential in nanostructure construction. Folic acid, composed of pterin, p-aminobenzoic acid and glutamic acid, exemplifies this trend with its self-assembly properties mediated through aromatic ring-driven stacking and nitrogen/oxygen atom interactions. Its hydrophilic groups, combined with its natural affinity for folate receptors on tumor cells, make fol