Conjugate Nanoparticles in Cancer Theranostics

Abstract

Nanotheranostics combines therapeutic and diagnostic functions within multifunctional nanoparticle platforms to enable precision medicine. This review outlines a comprehensive framework for engineering nanotheranostic systems, focusing on core material composition, surface functionalization, and stimuli-responsive drug delivery. Targeting strategies—from ligand-based recognition to biomimetic interfaces—are examined alongside therapeutic modalities such as chemotherapy, photothermal and photodynamic therapies, gene silencing via RNA interference, and radio sensitization. We discuss advanced imaging techniques (fluorescence imaging FI), magnetic resonance imaging (MRI), positron emission tomography (PET), and photoacoustic imaging for real-time tracking and treatment guidance. Key considerations include physicochemical characterization (e.g., article size, surface charge, and morphology), biocompatibility, in-vitro efficacy, and in-vivo biodistribution. We also address challenges such as rapid biological clearance, tumor heterogeneity, and clinical translation, and propose future directions for developing safe, adaptable, and effective nanotheranostic platforms. This review serves as a roadmap for advancing next-generation nano systems in biomedical applications.

1. Introduction

The convergence of nanotechnology and medicine has catalyzed the development of nanotheranostics—multifunctional nanoscale platforms that integrate both diagnostic imaging and therapeutic interventions. This emerging paradigm addresses key limitations of conventional disease management, such as off-target toxicity, suboptimal biodistribution, and limited imaging sensitivity, by enabling concurrent tracking, targeting, and treatment of diseased tissues [1,2,3]. At the foundation of nanotheranostics is the interplay between nanomaterial composition and surface functionalization, which determines physicochemical behavior, cellular uptake, and biological interactions [4,5,6,7]. A wide range of core materials—such as magnetic iron oxide [8], gold [9], and organic π-conjugated frameworks [10,11]—have been engineered as modular scaffolds to support drug delivery, imaging, and stimuli-responsive therapies. Here, the term “conjugate nanoparticles” refers specifically to nanocarriers functionalized via chemical conjugation with therapeutic or targeting ligands, rather than polymeric conjugated materials. Functionalizing these nanoplatforms with targeting ligands—including folic acid [2], arginyl-glycyl-aspartic acid (RGD) peptides [12], aptamers [13], and anti-EGFR conjugates [14]—further enhance tumor specificity and therapeutic efficacy.

Key design parameters—size, shape, surface charge, and stimulus-responsiveness—critically influence circulation half-life, tumor accumulation, and intracellular trafficking [15,16,17]. Optimization of these properties is essential to enhance therapeutic efficacy and diagnostic resolution. Analytical techniques such as transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), and magnetic relaxometry are instrumental in correlating nanostructure with function, enabling rational system optimization [18,19,20,21]. Advances in targeting strategies have significantly improved the selectivity and precision of nanotheranostic platforms. Passive targeting utilizes the enhanced permeability and retention (EPR) effect to promote tumor accumulation [22], while active targeting incorporates ligand–receptor interactions [8,23], pH- and redox-responsive systems [24,25], magnetic field-mediated navigation [26], and biomimetic coatings derived from immune or tumor cell membranes [27,28]. These strategies improve payload delivery to diseased sites, enhance cellular internalization, and enable spatially controlled therapeutic deployment. In addition, emerging closed-loop and feedback-responsive systems offer dynamic therapeutic control based on real-time physiological cues, representing a promising frontier in precision medicine.

Nanotheranostic systems are being designed for diverse therapeutic applications extending beyond conventional chemotherapeutics [29]. These include photothermal therapies [19,30,31], photodynamic therapy (PDT) [32,33], gene silencing via nucleic acid delivery [34], radiotherapy enhancement through nanocarrier-based radiosensitizers [35], and catalytic therapies exploiting tumor-associated redox imbalances [20]. This functional breadth facilitates synergistic multimodal treatments with minimized systemic toxicity. In parallel, integration of advanced imaging modalities—including FI [36], MRI [8], PET [28], photoacoustic imaging (PAI) [37], and ultrasound imaging [7]—enables real-time tracking, diagnosis, and intervention planning with high spatial and temporal resolution.

As nanotheranostic platforms progress toward clinical application, rigorous evaluation of safety and efficacy remains critical. In vitro studies validate cytotoxicity profiles and target specificity [13,18,33], while in vivo models assess biodistribution, clearance kinetics, and therapeutic outcomes [8,38,39]. These preclinical investigations provide foundational metrics for advancing translational potential and meeting regulatory requirements.

This review delivers a structured synthesis of nanotheranostics design principles—covering material selection, functionalization strategies, targeting mechanisms, imaging integration, and therapeutic applications—while also addressing major challenges in clinical translation, including biocompatibility, large-scale production, and regulatory considerations. Emphasis is placed on both preclinical development and clinical hurdles, with the aim of providing a clear, integrative framework for the rational design and future advancement of next-generation nanotheranostic systems.

2. Core Nanomaterial Composition and Functionalization

Nanomaterials for theranostic applications derive their effectiveness from the synergy between intrinsic core properties and versatile surface modifications. This dynamic interplay dictates the physicochemical behavior, biological interactions, and functional performance of nanoplatforms, enabling their precise operation within complex biological environments. However, considerations such as biodegradability and clinical scalability of core materials are increasingly critical for translational viability. This section systematically explores major classes of core nanomaterials and corresponding functionalization strategies that underpin the engineering of high-performance nanotheranostic systems.

2.1. Magnetic Nanoparticles

Magnetic nanoparticles—particularly iron oxide forms such as Fe₃O₄ and γ-Fe₂O₃—are extensively used in theranostics due to their superparamagnetic properties, enabling enhanced MRI contrast and magnetically guided drug delivery. To further augment imaging resolution, gadolinium-doped iron oxide (GdIO) nanoclusters have been developed to achieve dual T₁-T₂ MRI contrast, improving diagnostic sensitivity and tumor delineation [8].

Hybrid magnetic architectures introduce additional functionalities. For instance, Fe₃O₄-Au Janus nanoparticles, consisting of ~25 nm Fe₃O₄ and 9 nm Au domains, exhibit combined magnetic and plasmonic properties. These dual-surface nanoparticles enable simultaneous imaging and therapeutic delivery, with real-time intravital tracking of dye- or drug-conjugated payloads and enhanced T₂ relaxivity compared to clinical contrast agents [4].

Another advanced platform employs a core–shell–shell structure: Fe₃O₄@SiO₂@mesoporous SiO₂ nanoparticles, further modified with doxorubicin (DOX), gadolinium chelates like gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA), polyethylene glycol (PEG), and RGERPPR (RGE) peptides [arginine-glycine-glutamic acid-arginine-proline-proline-arginine] for tumor-specific targeting. This system demonstrated dual MRI contrast (r₁ = 6.13 mM⁻¹s⁻¹, r₂ = 36.89 mM⁻¹s⁻¹), pH-responsive drug release, and significantly enhanced uptake and apoptosis in U87MG glioblastoma cells both in-vitro and in-vivo, as shown in Figure 1 [5].

In particular, GdIO nanoclusters functionalized with cyclic arginine-glycine-aspartic acid-serine (cRGDs) peptides showed selective αvβ3 integrin targeting in Panc-1 pancreatic cancer models, supporting improved cytotoxicity, tumor-specific accumulation, and dual-mode imaging capability [8]. Despite their functional advantages, the persistence and potential long-term accumulation of metal oxide nanoparticles in vivo raise concerns regarding immune toxicity and clearance, limiting clinical progression. Collectively, these examples underline the versatility of magnetic nanoparticles in delivering integrated imaging, targeting, and therapeutic functions for precision oncology.

2.2. Gold-Based Nanostructures

Gold nanoparticles (AuNPs) are recognized for their chemical inertness, surface versatility, and tunable optical features—particularly in the near-infrared (NIR) region. Among advanced Au-based nanostructures, hollow gold nanorods (AuHNRs) are especially suited for photothermal therapy (PTT) in the NIR-II window due to their strong absorption at 1064 nm [9].

One system, AuHNRs-DTPP, combined AuHNRs with a photosensitizer-conjugated chimeric peptide (DTPP), enabling real-time apoptosis imaging and dual-mode NIR-II–responsive PTT/PDT. Upon 1064 nm laser exposure, the system released the photosensitizer in situ, supporting both heat-based ablation and reactive oxygen species–induced cytotoxicity, while minimizing off-target phototoxicity [9].

Separately, a multifunctional optical–magnetic nanoplatform was designed by integrating Au nanorods with mesoporous silica, gadolinium oxide, PEG, and folate via multiscale nano assembly. This construct exhibited MRI relaxivity up to 8-fold higher than clinical agents, supported two-photon imaging, and achieved targeted photothermal ablation of 10–12 mm tumors with low systemic toxicity and favorable clearance properties in vivo [40]. These AuHNRs-based platforms exemplify the potential of gold nanostructures to unify multimodal imaging, phototherapy, and biosafety for precision oncological applications. Nonetheless, gold-based nanostructures face scalability and cost challenges for large-scale production, and their non-biodegradable nature necessitates long-term biosafety evaluations.

2.3. Silica-Based Platforms

Mesoporous silica nanoparticles (MSNs) provide high surface area, tunable porosity, and excellent chemical stability, making them highly adaptable for drug delivery and imaging. Although silica is generally considered biocompatible, its slow degradation and potential for long-term accumulation warrant further in vivo safety profiling. Functionalized IBN-4 MSNs incorporating horseradish peroxidase (HRP) enabled enzyme–prodrug therapy and apoptosis induction in colon cancer models, with maintained catalytic activity for over 60 days and repeated usability [18]. Layered constructs like Fe₃O₄@SiO₂@mesoporous SiO₂ further integrate MRI contrast agents and tumor-targeting peptides for precise imaging and controlled release. A representative system—Fe₃O₄@SiO₂@mSiO₂/DOX-(Gd-DTPA)-PEG-RGE—showed pH-sensitive drug release, dual T₁/T₂ MRI contrast (r₁ = 6.13 mM⁻¹s⁻¹, r₂ = 36.89 mM⁻¹s⁻¹), and enhanced cytotoxicity in U87MG glioma cells, with in vivo studies confirming tumor accumulation and apoptosis [5]. In phototherapy, silica encapsulation improves dye retention and optical confinement. For example, gold nanorods coated with silica and loaded with indocyanine green (GNR@SiO₂-ICG) enabled fluorescence and two-photon luminescence imaging, while a single-wavelength CW laser triggered synergistic PPT and PDT, leading to effective A375 melanoma ablation with minimized dye leakage [41].

2.4. Carbon-Based Nanomaterials

Carbon-based nanostructures—including carbon quantum dots (CQDs), graphene oxide (GO), and multi-walled carbon nanotubes (MWCNTs)—are utilized for their fluorescence, conductivity, and biocompatibility. CQDs synthesized from almond resin and stabilized with honey exhibited a high quantum yield (61%) and minimal cytotoxicity, enabling multicolor FI and nuclear visualization with deep blue emission under 350 nm excitation [42]. PEGylated GO carriers were employed for the co-delivery of indocyanine green (ICG) and paclitaxel (PTX), supporting NIR imaging and synergistic chemotherapy. This multifunctional nanoplatform (NGO-PEG-ICG/PTX) demonstrated high loading efficiencies (ICG: 50 ± 2.1%, PTX: 90 ± 1.6%), strong tumor accumulation (29.1% ID/g at 24 h), extended retention (>8 days), and complete tumor suppression in-vivo without relapse or systemic toxicity following dual injections [43]. However, certain carbon-based materials—particularly MWCNTs—have raised concerns regarding immune activation, fibrotic responses, and long-term tissue persistence, posing translational limitations despite their functional promise.

2.5. Organic π-Conjugated and Polymer-Based Nanoparticles

Organic π-conjugated molecules—such as the fluorinated indacenodithiophene-based IDIC-4F [Indaceno [1,2-b:5,6-b′]dithiophene-2,7-diyl bis(difluorobenzylidene)malononitrile] [11] and spiro acridine derivative SFA-BTM [10H-spiro(acridine-9,9′-fluorene)] [10]—exhibit strong NIR-II absorption and high photothermal conversion efficiency, enabling carrier-free, self-assembling nanostructures with substantial therapeutic payload. Polymeric systems based on poly(lactide-co-glycolide) (PLGA) or polyfluorene derivatives provide structural and optical versatility for drug and imaging agent encapsulation [6,7]. For instance, perfluoropolyether-dopamine (PF-DPA) nanoparticles demonstrated dual-state fluorescence and cancer-selective cytotoxicity [6]. A PLGA-based platform co-loaded with IR825 (near-infrared dye), and bevacizumab enabled mitochondria-targeted photothermal therapy and multimodal imaging in thyroid cancer [7]. Meanwhile, both SFA-BTM and carrier-free IDIC-4F nanoparticles (DCF-P) exhibited potent photothermal activity, NIR-II fluorescence, and tumor ablation with high biocompatibility [10,11]. Importantly, many polymeric and organic nanomaterials—such as PLGA—are biodegradable and scalable under Good Manufacturing Practice (GMP) conditions, supporting their advancement toward clinical translation.

3. Surface Functionalization Strategies

3.1. PEGylation and Polymer Coatings

PEGylation is a cornerstone of nanocarrier engineering, improving aqueous solubility, reducing immunogenicity, and extending circulation time. PEGylation also reduces immune activation by limiting opsonization and complement system engagement, thereby enhancing systemic tolerance. For example, PEGylated oxidized alginate (mPEG-OAL) crosslinked with fluorescent carbon dots created nanoparticles capable of pH-triggered doxorubicin release and imaging-guided therapy [44]. Separately, β-cyclodextrin–functionalized magnetic nanoclusters enhanced MRI contrast, targeted delivery, and paclitaxel release [45], illustrating the synergistic impact of polymer coatings and host–guest interactions. Such polymer-based functionalization approaches are often GMP-compatible and scalable, making them favorable for clinical translation.

3.2. Targeting Ligands

Ligand conjugation enables receptor-specific targeting, enhancing selectivity and therapeutic outcomes. Folic acid (FA) facilitates tumor targeting in FA-receptor-overexpressing cancers, as seen in magnetic nanoparticles [2], graphene QDs [23], and carbon dots [46]. RGD peptides, targeting integrin αvβ3, improved delivery and imaging in glioblastoma and pancreatic cancer models [8,12]. Aptamers like epithelial cellular adhesion molecule (EpCAM) [1] and a 26-base guanine-rich oligodeoxyribonucleotide that binds to nucleolin (AS1411), support receptor-mediated uptake and tumor-specific delivery. However, the chemical conjugation of targeting ligands can introduce batch-to-batch variability, necessitating stringent quality control measures during manufacturing.

An illustrative example is shown in Figure 2, which presents an anionic linear globular dendrimer G2 (ALGDG2) system conjugated with the AS1411 aptamer to deliver Iohexol, a CT contrast agent, specifically to MCF-7 breast cancer cells. This platform demonstrated targeted delivery, reduced cytotoxicity to normal cells, and enhanced imaging specificity, highlighting aptamer-based targeting as a precise and effective approach [13], supporting selective imaging and therapy in colorectal and breast cancers. Monoclonal antibodies such as cetuximab and trastuzumab enabled epidermal growth factor receptor (EGFR)- and human epidermal growth factor receptor 2 (HER2)-targeted imaging and drug delivery across multiple nanoplatforms [14,47,48]. Antibody-functionalized systems, while highly specific, may face challenges in reproducibility and scalability due to the complexity of bioconjugation protocols.

3.3. Stimuli-Responsive Moieties

Stimuli-responsive linkers improve therapeutic precision by enabling on-demand drug release. Redox-sensitive disulfide bonds, used in glutathione-activated pseudopolyrotaxane carriers, support tumor-specific PDT [25]. pH-sensitive constructs like iron oxide, methyl methacrylate and doxorubicin (IO-MMA-DOX) utilize hydrazone bonds for acid-triggered release with MRI-guided targeting [17]. Enzyme-sensitive aggregation-induced emission luminogen (AIEgen)–peptide conjugates respond to proteases such as cathepsins, inducing intracellular condensation for sustained imaging and photodynamic efficacy [32]. These stimuli-responsive mechanisms may also reduce off-target immune activation by confining drug activity to tumor-specific microenvironments.

3.4. Fluorescent and Imaging Enhancers

Integrating contrast agents into nanocarriers enables multimodal imaging. Gadolinium(III) complex functionalized with a boron-dipyrromethene derivative [BODIPY–Gd(III)] nano discs supported photoacoustic and MR imaging alongside photothermal therapy [37]. Gadolinium(III) oxide and copper(II) sulfide (Gd₂O₃/CuS) nanodots synthesized via albumin nanoreactors combined T₁-weighted MRI with NIR FI and photothermal ablation [49]. Some imaging functionalization, especially those incorporating clinically approved agents like gadolinium, enable GMP-compliant synthesis routes, thereby streamlining the regulatory approval process.

3.5. Biomimetic and Bioinspired Interfaces

Biomimetic coatings enhance immune evasion and targeting. Cancer cell membrane–derived liposomes functionalized with porphyrin and ⁸⁹Zr enabled homotypic targeting, PET imaging, and PDT [28]. Albumin-based systems further improved delivery and imaging, exemplified by albumin-coated Gd₂O₃/CuS nanodots [49] and Evans blue (EB)–camptothecin (CPT) conjugates used in clinical imaging and treatment [35]. These bioinspired interfaces reduce complement activation and macrophage uptake by mimicking native biological surfaces, enhancing circulation and tumor accumulation.

The integration of nanoparticle core design and surface functionalization is central to effective theranostic performance. Hybrid nanoplatforms—such as Gd-DTPA and peptide-modified Fe₃O₄@SiO₂@mesoporous SiO₂ systems—achieve dual MRI contrast, pH-responsive release, and enhanced tumor uptake [5]. Ligand-directed platforms employing FA, RGD, aptamers, or antibodies allow precise receptor-mediated delivery and imaging [1,8,14]. Stimuli-responsive architectures—such as acid-cleavable iron oxide–doxorubicin systems [17], glutathione-sensitive carriers [25], and pH-triggered GO-based nanohybrids [43]—exemplify the tailored design approaches enabling next-generation, precision-targeted nanomedicine. Scalability of these platforms varies by strategy, with simpler chemical conjugations and polymer coatings generally offering more robust and GMP-compatible production routes than complex biomimetic or antibody-based systems.

Table 1. Nanomaterial Platforms: Core Structures, Functionalization Strategies, and Biomedical Applications.

Nanoplatform Class	Structural Features and Surface Engineering	Functional Role in Theranostics	Clinical Status/Challenges/Limitations	Supporting Studies
Magnetic Iron-Based Nanoparticles	Magnetic cores (Fe3O4, γ-Fe2O3, MnFe2O4, CoFe2O4, Fe/Fe3O4) combined with PEG, PDA, or antibody/ligand coatings (e.g., RGD, FA) enable tunable magnetism, colloidal stability, and targeted delivery.	MRI contrast agents, magnetothermal therapy, pH-sensitive drug release	Preclinical; effective imaging and delivery. Challenges: toxicity (e.g., Co), clearance, scale-up, and in vivo consistency.	[2,5,15,17,45,50,51,52]
Gold-Based Nanomaterials	Au cores (solid, hollow, Janus) functionalized with PEG, antibodies, or photo agents (e.g., ICG, Ce6) allow surface plasmon resonance-based imaging and photothermal conversion.	NIR-triggered phototherapy, photoacoustic imaging, theranostics	Preclinical; strong photothermal effects. Issues: size optimization, organ accumulation, gold persistence, and biocompatibility.	[4,9,14,47,53,54]
Gadolinium & Lanthanide MRI Agents	Gd3+ or Yb3+ cores encapsulated in polymers or conjugated to dendrimers, PEG, or targeting ligands (FA, RGD) enable high relaxivity, biocompatibility, and dual-modality imaging.	MRI contrast, tumor tracking, multimodal diagnosis	Preclinical; high relaxivity and targeting. Limits: Gd3+ toxicity, complex synthesis, and long-term safety unknown.	[23,55,56,57,58,59]
Targeting Ligands (RGD, FA, Antibodies, Aptamers)	Diverse nanocores (metallic, polymeric, QDs) are functionalized with specific ligands (RGD, FA, EGFR, aptamers) to match biological receptors, boosting cell-type specificity.	Receptor-mediated uptake, blood–brain barrier (BBB) penetration, cancer targeting	Preclinical; improved uptake and specificity. Hurdles: ligand stability, immune responses, receptor heterogeneity.	[2,14,15,23,53,54,60,61]
Polymer & Micelle Nanocarriers	Amphiphilic polymers (PLGA, PEG, PAA, TPGS) self-assemble around hydrophobic cores or drugs, with surface modification (antibodies, peptides) enhancing stability and controlled release.	Stimuli-responsive chemotherapy, combinational drug delivery	Preclinical; controlled release and targeting shown. Barriers: synthesis reproducibility, degradation, and scale-up.	[7,39,62,63,64,65,66]
Quantum Dots (Carbon, Graphene, Cu-In-S/ZnS)	Fluorescent cores (CQDs, GQDs, CuInS/ZnS) are surface engineered with PEG, FA, or aptamers, providing water solubility, charge balance, and targeted imaging properties.	Bioimaging, targeted drug delivery, light-activated therapy	Preclinical; strong imaging and delivery. Challenges: metal toxicity, photostability, biosafety, and synthesis control.	[1,23,46,67,68,69]
Photosensitizers & Photothermal Agents	Organic/inorganic cores (ICG, Ce6, porphyrins, BODIPY, π-conjugates) functionalized via covalent or cleavable bonds enhance solubility, targeting, and light-triggered activation.	Photothermal therapy, PDT, NIRII diagnostics	Preclinical; potent ablation and imaging. Issues: light penetration, ROS side effects, retention, and targeting control.	[9,10,25,37,41,60,70]
Self-Assembled & Carrier-Free Nanodrugs	Drugs or conjugates (e.g., PTX, CPT, IDIC-4F) self-assemble into nanostructures via π–π or H-bond interactions; targeting or responsive units (e.g., disulfides) enhance delivery precision.	High drug loading, redox-sensitive delivery, structure-defined release	Some early clinical data (e.g., EB-CPT). Generally preclinical. Key issues: safety, synthesis, and release kinetics.	[11,35,36,71,72,73]
Hybrid/Core–Shell Nanostructures	Multi-core architectures (e.g., Fe3O4@SiO2@Au, QDs@HA) functionalized with polymers or biomolecules for combining magnetic, optical, and biochemical responses.	Dual-mode imaging, on-demand drug release, spatiotemporal control	Preclinical; multimodal functions. Obstacles: complex fabrication, stability, clearance, and multi-material regulations.	[4,29,34,40,52,74,75]
Biologically Derived/Biomimetic Systems	Natural or cell membrane-based nanocarriers (RBC, NK, cancer cell, HDL) function with therapeutic agents or imaging dyes retain surface proteins to enhance targeting and immune evasion.	Biomimetic delivery, prolonged circulation, immune escape	Preclinical; strong targeting and circulation. Challenges: membrane source consistency, immune response, scalability.	[27,28,76,77,78]
Radiolabeled & Multimodal Theranostics	Nanocores (liposomes, polymers, AuNPs) radiolabeled with isotopes (99mTc, 89Zr, 131I) and functionalized with ligands (FA, PEG, antibody) for concurrent imaging and therapy.	PET/SPECT-guided delivery, radiotherapy, image-based treatment planning	Some clinical translation (e.g., EB-CPT). Most are preclinical. Key concerns: radiolabel stability, handling, regulation.	[22,28,35,39,79]

summarizes diverse nanomaterial platforms, demonstrating how core–shell design and functional surface adaptation align with specific biomedical goals. Scheme 1 provides a conceptual schematic, mapping the integration of key modules—core materials, targeting ligands, responsive linkers, and imaging agents—into unified nanotheranostic systems.

4. Physicochemical Profiling of Nanoparticles

Comprehensive physicochemical characterization is a cornerstone of rational nanotheranostics design, as these properties directly influence biological behavior—governing circulation, biodistribution, targeting accuracy, therapeutic release, and imaging performance. In parallel, practical considerations such as shelf life, storage stability, and batch-to-batch reproducibility are critical for clinical and commercial translation. This section outlines the key physicochemical parameters and primary analytical techniques used to evaluate nanotheranostics platforms, while also addressing strategies to maintain colloidal stability, surface functionality, and payload integrity over time. Methods such as lyophilization with cryoprotectants, inert atmosphere storage, and pH-controlled formulations are discussed, alongside the importance of reproducibility in synthesis—validated through consistent particle size, zeta potential, and drug loading—to meet regulatory standards and ensure therapeutic reliability.

4.1. Particle Size and Morphology: Defining Biological Fate

Nanoparticle size and shape dictate their biological interactions, influencing systemic circulation, cellular uptake, tumor penetration, and clearance routes. Particles in the 10–200 nm range are ideal for tumor accumulation via the EPR effect, while ultrasmall particles (<10 nm) support renal clearance and enhanced interstitial diffusion.

TEM and DLS are commonly employed to assess morphology and hydrodynamic size. Fe₃O₄-Au Janus nanohybrids, for example, displayed a ~25 nm magnetite and 9 nm gold dual-surface Janus configuration, enabling combined MRI, drug delivery, and real-time tumor tracking [4]. Superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with folic acid showed size expansion to 67 nm after DOX loading, confirming successful drug encapsulation. The resulting formulation offered pH-sensitive drug release, enhanced tumor uptake, and MRI-guided therapy [15]. Ultrasmall, carrier-free IDIC-4F nanoparticles (~4 nm) exhibited efficient tumor penetration, renal clearance, and high photothermal conversion efficiency (80.5%), affirming their potential for next-generation theranostics [11].

4.2. Surface Charge (Zeta Potential): Indicator of Stability and Cellular Interaction

Zeta potential is a key determinant of colloidal stability, cellular uptake, and circulation behavior. Nanoparticles with moderately negative zeta potentials (−10 to −30 mV) generally display reduced aggregation and prolonged systemic persistence.

Terbium-doped carbon dots (CD-Tb) with a zeta potential of −20.7 mV maintained stable dispersion and high DOX loading (92.5%), achieving superior cytotoxicity against colon cancer cells compared to free DOX [16]. PEGylation neutralizes surface charge, improving biocompatibility and systemic stability. PEG-coated Fe₃O₄-Au hybrids demonstrated enhanced MRI relaxivity, photothermal conversion, and complete tumor ablation within six days [19]. In contrast, dextran- or chitosan-coated ferrite particles showed increased aggregation and reduced colloidal stability, despite notable magnetic hyperthermia performance, highlighting PEG’s advantages for intravenous use [21]. Zeta potential measurements have also been used to monitor colloidal stability over time, confirming minimal aggregation in stored formulations.

4.3. Drug Loading and Controlled Release: Efficacy with Precision

High drug loading and stimulus-responsive release enhance treatment selectivity and minimize systemic toxicity. Nanocarriers are engineered to respond to tumor microenvironmental triggers such as low pH, redox gradients, or high intracellular glutathione (GSH) levels.

CD-Tb nanoparticles achieved 92.5% DOX loading and 89% pH-sensitive release, significantly outperforming free DOX in cytotoxicity assays [16]. Iron oxide–based carriers exhibited pH-triggered DOX release, MRI contrast, and folate receptor–mediated uptake [17]. Redox-sensitive platforms—such as a cyclen–camptothecin conjugate—enabled GSH-triggered release alongside dual-mode imaging [58]. A dopamine-linked, anti-VEGF-functionalized SPION system conjugated to bortezomib achieved targeted, imaging-guided therapy in liver cancer [51]. Carrier-free IDIC-4F nanostructures offered near-complete drug incorporation, high photothermal conversion efficiency, and effective dual-mode imaging and therapy [11]. Drug retention during storage is routinely evaluated to confirm loading stability and prevent premature leakage under non-physiological conditions.

4.4. Magnetic and Relaxometric Properties: MRI and Hyperthermia Readiness

Magnetic responsiveness and T₁/T₂ relaxivity define MRI contrast capability and suitability for magnetic hyperthermia. High-saturation magnetization Fe₃O₄ nanoparticles (~92 emu/g), conjugated to DOX, enabled magnetothermal chemotherapy and MRI-guided delivery, achieving ~96% cancer cell death within 30 min of exposure [50].

Ultrasmall Gd₂O₃ nanoparticles functionalized with lactoferrin and RGD₂ peptides achieved a T₁ relaxivity of 60.8 mM⁻¹·s⁻¹ and ΔSNR of ~423% in glioblastoma imaging, while effectively penetrating the BBB [56]. A chitosan-based SPION/cerium oxide composite demonstrated T₂ relaxivity of 409.5 mM⁻¹·s⁻¹, enabled ROS scavenging, methotrexate delivery, and significant tumor suppression without recurrence [20]. Repeat relaxivity measurements across stored batches have confirmed reproducibility in imaging performance and magnetic response.

4.5. Surface Functionalization and Ligand Conjugation: Enhancing Target Specificity

Surface ligands direct nanoparticles toward specific cellular receptors, increasing therapeutic precision and imaging specificity. Functionalization is validated using FTIR, UV-Vis spectrophotometry, and fluorescence-based techniques.

MWCNTs conjugated with Mucin-1 (MUC-1) aptamers showed targeted uptake in MDA-MB-231 (MD Anderson metastatic breast cancer cell line #231) cells, effective photothermal ablation, and reduced inflammatory markers. This construct also served as an MRI contrast agent [80]. RGD-modified gold dendrimers achieved integrin-targeted computed tomography (CT) contrast and induced ROS-mediated apoptosis in tumor cell models [54]. Folate-functionalized Fe₃O₄ nanoparticles with tuned ligand valency achieved differential tumor cell binding, enhanced MRI signals, and validated targeting efficacy in folate receptor (FR)-expressing KB and HeLa models [81]. Long-term preservation of ligand-binding activity has been demonstrated in nanoparticles stored under inert atmospheres or at low temperatures, indicating sustained targeting functionality and overall formulation stability.

4.6. Optical and Photothermal Properties: Imaging and Therapy Integration

Photothermal conversion efficiency (PCE), quantum yield, and fluorescence stability govern the utility of nanoparticles in optical imaging and phototherapy.

Triplet tellurophene-based PNDI-2T (a semiconducting polymer nanoparticle incorporating tellurophene for enhanced triplet state generation) exhibited ~45% PCE, strong NIR absorbance, and high ROS yield, making it ideal for imaging-guided photothermal and PDT [4]. Gd₂O₃/CuS nanodots synthesized via an albumin nanoreactor achieved a PCE of 45.5%, high T₁ relaxivity, and NIR fluorescence for effective tumor ablation [49]. CQDs prepared through green synthesis using almond resin demonstrated a 61% quantum yield, high photostability, and biocompatibility, enabling multicolor cellular imaging [42]. CD-Tb particles supported high drug-loading capacity and cytotoxic efficacy while retaining functional integrity under biological conditions [16]. Fluorescence intensity and photothermal conversion efficiency are routinely monitored during storage to validate optical stability and reproducibility.

4.7. Structural and Elemental Validation: Confirming Material Integrity

Structural integrity and elemental composition are validated through X-ray diffraction (XRD), energy-dispersive spectroscopy (EDS), Brunauer–Emmett–Teller (BET) analysis, and thermal analysis methods. These confirm crystallinity, element distribution, porosity, and thermal stability—critical for storage and in-vivo performance.

BET analysis has been applied to magnetic fiber composites to determine surface area relevant to drug loading and release kinetics [82]. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) confirm thermal resilience in polymeric platforms [82]. For instance, gold nanorod–based probes incorporating mesoporous silica, gadolinium oxide, and folate-PEG underwent computational nano assembly and structural validation to demonstrate multifunctional performance and tumor-targeted imaging [40]. These methods also support shelf-life evaluation by detecting degradation or compositional changes under varied storage conditions.

4.8. Stability and Biocompatibility: Ensuring Functional Performance

Nanoparticles must retain stability under physiological conditions and demonstrate biocompatibility to progress toward clinical application. This includes preserving colloidal, chemical, and thermal integrity while resisting degradation or aggregation.

RGD- and α-tocopheryl succinate–functionalized gold dendrimer nanoparticles remained stable across pH 5–8 and temperatures from 4 °C to 50 °C, while retaining ROS-generating capacity [54]. HDL-inspired biomimetic nanoparticles incorporating ICG and iRGD peptide demonstrated high photostability, ROS production under NIR irradiation, and in-vivo structural integrity—confirming their utility in photo-theranostics [77]. Accelerated stability testing and storage under stress conditions (e.g., elevated temperature or light exposure) have been used to assess shelf life and predict long-term performance.

Comprehensive physicochemical profiling—including assessments of size, zeta potential, ligand targeting, imaging relaxivity, drug release behavior, and environmental stability—is essential for validating nanotheranostic platforms. Systems such as Fe₃O₄-Au Janus hybrids [4], CD-Tb carbon dots [16], redox-sensitive IONP constructs [17], semiconducting polymer nanoparticles [31], and RGD-functionalized MRI-visible platforms [54,56] exemplify this approach. Incorporating shelf life, reproducibility, and storage assessments further enhances translational feasibility. Such profiling not only ensures therapeutic and diagnostic efficacy but also supports translational development and regulatory readiness.

5. Mechanisms of Drug Loading and Release

The therapeutic efficacy of nanomedicine depends heavily on the ability of nanocarriers to deliver drugs in a controlled, targeted, and biocompatible manner. Advanced drug loading and release strategies are designed to ensure spatial and temporal precision, reduce systemic toxicity, and optimize therapeutic outcomes. These strategies are dictated by nanoplatform design and exploit tumor-specific conditions or externally applied stimuli. Emerging approaches now incorporate spatiotemporal and feedback-controlled systems, which adjust drug release dynamically in response to local physiological cues, enhancing precision in heterogeneous tumor microenvironments. This section delineates the principal mechanisms employed for drug incorporation and release in contemporary nanotheranostic systems.

5.1. pH-Responsive Drug Release

Tumor environments (pH~6.5–6.8), along with acidic intracellular compartments like endosomes and lysosomes (pH~4.5–6.0), provide an exploitable differential for pH-sensitive drug release. Such responsiveness allows nanocarriers to release therapeutic payloads selectively at tumor sites, minimizing off-target toxicity.

For example, folic acid–modified carbon dot nanocarriers disassembled in acidic environments to release drugs within folate receptor–positive tumors [46]. Likewise, SPIONs functionalized with folic acid (DOX@FA-SPIONs) exhibited pH-responsive doxorubicin release, enhanced MRI contrast (r₂ = 81.77 mM⁻¹s⁻¹), and active targeting via folate receptors [15]. This system is visualized in Figure 3, which schematically illustrates both the synthesis of FA-modified SPIONs (Figure 3a) and their functional role in magnetically guided, pH-sensitive drug delivery (Figure 3b). The Fe₃O₄ core provides T₂ MRI contrast and magnetic targeting, FA promotes receptor-mediated uptake in cancer cells, while other coating components stabilize the nanocarrier. By contrast, systems like poly(L-γ-glutamyl-glutamine)–paclitaxel conjugates have shown promising tumor accumulation guided by MRI, but their pH-dependent release remains insufficiently demonstrated [55].

5.2. Redox-Responsive and GSH-Triggered Release

Elevated intracellular GSH levels in cancer cells (up to 2–10 mM) create a reducing environment suitable for redox-responsive drug release, typically facilitated through disulfide bond cleavage or other redox-labile linkers. Dual pH/GSH-responsive nanoplatforms composed of hollow mesoporous silica, carbon dots, and DOX have demonstrated efficient intracellular drug release and enhanced tumor suppression through combined imaging and therapy [24]. Lanthanide-camptothecin nanocomposites further exemplify this approach, achieving GSH-triggered CPT release while enabling dual-mode NIR/MR imaging [58]. In contrast, systems such as the capecitabine-loaded ROS-sensitive micelles [83] are not truly GSH-triggered and are more accurately categorized under oxidative or pH-responsive mechanisms.

A theranostic platform comprising NIR-emitting carbon dots, hollow mesoporous silica nanoparticles, and pH/GSH-responsive polylysine was developed for drug delivery. DOX was loaded and released in tumor cells via reduction of disulfide bonds and acidic pH-triggered charge conversion. In vitro and in vivo studies showed selective tumor accumulation and minimized side effects. Drug loading capacity: 15.3 wt%; cumulative DOX release at pH 6.5 with GSH: ~82% after 48 h [24].

5.3. Prodrug Strategies: Covalent Conjugation and Stimuli-Cleavable Linkers

Prodrug strategies involve the covalent attachment of therapeutics to nanocarriers through cleavable linkers such as disulfide, ester, or hydrazone bonds. These linkers are responsive to tumor-specific stimuli, enabling localized drug activation. For example, a reduction-activatable tetrameric paclitaxel prodrug conjugated with a porphyrin photosensitizer formed redox-sensitive nanoparticles that released paclitaxel under intracellular reducing conditions while facilitating photodynamic imaging through the porphyrin core [72]. Doxorubicin-loaded polymeric nanoparticles combined with in situ–formed gold nanoparticles have also demonstrated GSH- and pH-triggered fluorescence recovery and tumor-selective cytotoxicity, enabling real-time imaging-guided therapy in MCF-7 (Michigan Cancer Foundation-7) models [74].

5.4. Physical Encapsulation and Sustained Release

Physical encapsulation strategies utilize non-covalent interactions such as hydrophobic forces, hydrogen bonding, or electrostatics for drug loading. Although generally non-responsive to biological stimuli, these systems support sustained release and passive tumor targeting via the EPR effect. Polyfluorene-based PF-HQ (fluorescent hydroxyquinoline-affixed polyfluorene) nanoparticles, for instance, physically encapsulated DOX to achieve tumor accumulation, enable FI, and facilitate therapeutic monitoring in melanoma models [62]. Moreover, mesoporous silica–hybrid platforms incorporating QDs and gold nanoparticles effectively encapsulated epirubicin for targeted colorectal tumor delivery and imaging-guided chemo-radiotherapy [1]. Systems like DOX@FA-SPIONs also involve physical encapsulation; however, due to their pH-responsive release and magnetic targeting, they should be more accurately classified as hybrid platforms [15].

5.5. Light-, Heat-, and Ultrasound-Triggered Release

External stimuli such as NIR light, heat, and ultrasound provide non-invasive, spatiotemporally controlled release. These modalities are valuable in integrating therapy and diagnostics. For instance, upconversion nanoparticle (UCNP)-based nanofibers, loaded with DOX, achieved photothermal conversion (~62.7 °C) under NIR irradiation, triggering drug release and reactive oxygen species (ROS)-induced apoptosis in breast cancer [84]. Fe₃O₄-Au Janus nanohybrids offered dual benefits of magnetic targeting and laser-induced heat-triggered release, with real-time intravital microscopy monitoring [4]. On-demand release platforms, activated by external signals such as light or ultrasound, enable precision dosing and dynamic therapeutic control, particularly in responsive or relapse-prone tumor microenvironments.

A particularly well-integrated example is shown in Figure 4, where ultrasound-responsive (Dex@NBs-TRPC6) nanobubbles were developed for targeted delivery of dexamethasone to renal tissues. The nanobubbles used TRPC6 antibodies for active targeting of glomerular podocytes, while ultrasound facilitated both drug release and imaging. This system effectively treated adriamycin-induced nephropathy, demonstrating the utility of externally triggered delivery in non-cancer applications [85].

5.6. Biochemical Triggering: Enzyme- and miRNA-Responsive Systems

Endogenous biomarkers such as overexpressed enzymes and dysregulated microRNAs (miRNAs) offer selective internal triggers for drug release, enhancing tumor specificity. A miRNA-21–responsive nanohybrid composed of Fe₃O₄@polydopamine with a DOX-DNA corona enabled targeted release, magnetic guidance, gene silencing, and photothermal therapy, resulting in substantial tumor growth inhibition and extended survival [34]. Likewise, a DNA nanotriangle–based multivalent aptamer probe (NTri-SAAP) underwent conformational change upon recognition of cell surface biomarkers, selectively releasing DOX and achieving over 80% tumor inhibition in-vivo [86]. While miRNA- and aptamer-responsive systems are emphasized here, enzyme-activated nanocarriers—targeting cathepsins, matrix metalloproteinases (MMPs), or esterases—also offer substantial specificity and should be considered part of a comprehensive biochemical targeting strategy.

5.7. Radiolabeled Drug Delivery and Internal Radiation Triggers

Radiolabeled or boron-conjugated nanocarriers that integrate therapeutic agents with imaging or radiation-responsive elements offer dual advantages: real-time tracking and localized cytotoxicity through neutron or radiation-enhanced mechanisms. For instance, an albumin–gemcitabine–closo-dodecaborate conjugate used a pH-sensitive linker for tumor-selective release while enabling FI and integration with boron neutron capture therapy (BNCT) [87]. Likewise, Gd₃N@C₈₀-based liposomes co-loaded with doxorubicin supported MR imaging and dual-receptor targeting of glioblastoma, demonstrating the potential of metallofullerene-enhanced theranostics [57]. Although direct radiation-triggered release mechanisms—such as radiolysis or decay-induced linker cleavage—are still in the early stages of development, existing platforms often combine therapeutic delivery with imaging and traditional stimuli-responsive strategies to improve precision.

Advanced drug loading and release mechanisms are fundamental to the success of nanotheranostic systems. These strategies—ranging from pH- and redox-responsiveness to prodrug activation, external stimuli, biochemical cues, and radiolabel integration—provide multi-tiered control over therapeutic delivery. By tailoring release mechanisms to tumor-specific environments or applying non-invasive external triggers, nanocarriers are able to maximize therapeutic precision while minimizing systemic toxicity. Incorporating feedback-controlled and on-demand release technologies further enhance adaptability, offering personalized and temporally optimized drug administration. Continued innovation in these domains is expected to drive the clinical translation of next-generation precision nanomedicine platforms.

Table 2. Stimuli-Responsive Nanocarrier Systems for Controlled Drug Release and Activation.

Stimulus Type	Mode of Action	Implementation Considerations	Selected Literature
pH-Responsive Systems	Acidic tumor or endosomal pH triggers drug release (e.g., DOX, PTX); in some systems, pH also promotes endosomal escape or conformation change for cellular uptake.	Requires careful pKa tuning to avoid premature leakage; endosomal escape mechanisms (e.g., proton sponge effect) can be integrated for enhanced cytosolic delivery. Preclinical (in vitro/in vivo, no human trials).	[2,5,24,43,51,68,69,88]
Redox-Responsive Systems (GSH-Sensitive)	High intracellular GSH cleaves disulfide or selenoether bonds, enabling drug release inside tumor cells; redox environment may also enable structural transformation for targeting or cell entry.	Ideal for cytosolic drug delivery; linker length and steric affect cleavage efficiency; combination with other stimuli can improve selectivity. Preclinical only.	[24,25,36,58]
Light/NIR-Triggered Systems	Light (especially NIR) is used to trigger drug release or photoactivation (PDT/PTT); in some designs, it also activates targeting via conformation switch or cleavage of a masking group.	Allows precise control of activation site and timing; requires device integration and tumor accessibility; suitable for superficial tumors or guided fiber delivery. Evaluated only in mice.	[7,9,84,89]
Enzyme-Responsive Systems	Tumor-specific enzymes (e.g., MMPs, cathepsins) cleave linkers to trigger drug release or activate targeting ligands (e.g., PEG shedding exposes targeting domain).	Enzyme specificity can reduce off-target effects; co-expression variability across tumor types requires careful biomarker selection and validation. Preclinical efficacy demonstrated.	[32]
MicroRNA or mRNA-Activated Systems	Tumor-overexpressed mRNA/miRNA activates DNA zymes or opens nanostructures for targeted release; in some cases, this also triggers exposure of targeting moieties.	Allows personalized therapy; requires sequence specificity and intracellular delivery to the cytosol; may be combined with nanocarriers enabling endosomal escape. Tested in vitro/in vivo.	[34,90]
Photothermal/Photodynamic Activation	Photothermal or photodynamic agents generate heat or ROS to induce cytotoxicity or rupture nanocarriers, releasing payload; can also aid membrane permeability or nuclear/mitochondrial targeting.	Good for drug-resistant tumors; ROS/heat must be tightly controlled to avoid off-target effects; photothermal conversion efficiency and stability are key design factors. All preclinical.	[3,22,28,31,66,91,92]
Tumor Microenvironment-Driven Passive Systems	Leverage endogenous features (e.g., low pH, high GSH, EPR effect) for passive targeting and drug release, often without external ligands.	Simpler synthesis and scalable; works better in highly vascularized tumors; limited control over precise targeting or timing, often combined with active or external triggers. In vivo only.	[24,33,36,58]
Biomimetic Activation (e.g., Cell Membrane Cloaking)	Biomimetic coatings can unmask targeting ligands or release drugs in response to tumor microenvironment cues, offering immune evasion and responsive targeting.	High biocompatibility and circulation time; batch variability and reproducibility are manufacturing challenges; responsive uncoating or degradation improves tumor homing. Shown effective in glioma models only.	[27]

summarizes the primary classes of stimuli-responsive systems, highlighting mechanisms, release conditions, and functional benefits. Scheme 2 provides a schematic overview of internal and external triggering strategies for advanced drug delivery in nanotheranostics applications.

6. Targeting Strategies in Nanomedicine

A defining advantage of nanomedicine lies in its capacity to direct therapeutic agents to pathological tissues while minimizing systemic toxicity. Targeting strategies have evolved to harness molecular recognition, tumor microenvironment cues, and external stimuli, thereby enhancing localization, retention, and therapeutic performance. Often deployed in tandem, these approaches include active targeting, passive accumulation, stimuli-responsive release, magnetic guidance, organelle-specific localization, biomimicry, and dual-targeting integrations. However, the efficacy of these strategies is often compromised by tumor heterogeneity, which results in variable receptor expression, uneven vascularization, and dynamic microenvironmental conditions that limit uniform nanoparticle delivery. This section outlines the major strategies and highlights key applications within precision nanomedicine.

6.1. Active Targeting via Ligand–Receptor Interactions

Active targeting involves the functionalization of nanoparticles with ligands that selectively bind to receptors overexpressed on diseased cells, thereby enhancing cellular uptake and improving therapeutic selectivity. Folic acid (FA) is widely used to target folate receptors, which are highly expressed in ovarian, breast, and colorectal cancers [17,88,93]. RGD peptides, known for their high affinity to integrin αvβ3, facilitate targeted delivery in aggressive tumors such as glioblastoma and triple-negative breast cancer [8,72,94]. EGFR targeting is accomplished using ligands such as cetuximab or anti-EGFR antibodies, which enhance nanoparticle delivery to breast and head and neck tumors [48,76]. Aptamers such as AS1411, which targets nucleolin, and protein tyrosine kinase 7 (PTK7)-specific sequences offer additional advantages, including high binding affinity, low immunogenicity, and efficient cellular recognition [1,13,53,95]. Transferrin and lactoferrin ligands are particularly effective for central nervous system targeting, as they facilitate BBB translocation [57,96]. Similarly, hyaluronic acid (HA) is frequently employed to target cluster of differentiation 44 (CD44) receptors, which are prevalent in many solid tumors [64,97,98]. To address inter- and intra-tumoral variability, personalized targeting approaches that tailor ligand selection to patient-specific biomarker profiles are increasingly under investigation.

6.2. Passive Targeting via the EPR Effect

Passive targeting capitalizes on the abnormal vasculature and deficient lymphatic drainage in tumors, leading to nanoparticle accumulation through the EPR effect. Nanoparticles of appropriate size (typically 10–200 nm) are particularly efficient in exploiting this mechanism. PEGylation is commonly utilized to extend circulation time and reduce immune system clearance, thereby supporting effective passive accumulation. Additionally, ultrasmall nanoparticle constructs are capable of improved tumor penetration and retention due to their rapid diffusion and partial renal clearance [92]. Albumin-based nanocarriers also offer a form of passive targeting by leveraging natural transport pathways and enhanced accumulation in tumors, functioning independently of specific receptor interactions [3,35,87]. Nonetheless, the heterogeneity of tumor vasculature can limit uniform EPR-based delivery, necessitating adaptive strategies that respond to the evolving tumor milieu.

6.3. Stimuli-Responsive Targeting

Stimuli-responsive nanocarriers are engineered to release therapeutic payloads in response to specific internal conditions of the tumor microenvironment, enhancing site-specific delivery. pH-sensitive systems utilize acid-labile linkers or charge-conversion mechanisms to release drugs in acidic tumor environments [33,82]. Redox-responsive systems are designed with disulfide or selenoether bonds that cleave in high-GSH conditions characteristic of tumor cells [24,36]. Carriers sensitive to dysregulated microRNAs like miRNA-21, or activated by specific enzymes such as HRP, enable biomarker-triggered release in tumor settings [34]. Furthermore, dual-responsive systems combine multiple triggers—such as pH and redox sensitivity—or incorporate both endogenous and external stimuli, such as NIR light, to achieve superior spatiotemporal control over drug release [25,99]. To counteract spatial and temporal variability within tumors, adaptive delivery systems capable of real-time environmental sensing are being developed to optimize payload release.

6.4. Magnetic Targeting

Magnetic targeting utilizes externally applied magnetic fields to direct superparamagnetic nanoparticles, such as iron oxide (Fe₃O₄), to tumor tissues. This approach is often integrated with receptor-mediated strategies to enhance specificity and precision. For example, RGD-conjugated magnetic particles have demonstrated targeted accumulation in tumors that overexpress integrins [26]. SPION-based nanocarriers can also be modified with nucleic acids or other bioactive elements to facilitate magnetically guided, gene-directed delivery [34,90].

6.5. Organelle-Targeted Delivery

Targeting specific intracellular organelles significantly enhances therapeutic efficacy by directing drugs to critical cellular compartments. Mitochondria-targeting nanoplatforms—such as IR825-loaded systems—enable potent photothermal and photodynamic effects upon NIR light exposure, resulting in mitochondrial disruption and apoptosis [7,66,71]. For instance, TNPT, a mitochondria-targeted AIE-cisplatin conjugate, demonstrated 2.4× greater potency than cisplatin and superior ROS generation under light exposure [71]. Similarly, lysosome-targeted nanocarriers like HA–Mn₂O₃/HCQ hybrids facilitate lysosomal accumulation, pH modulation, and cytoplasmic escape, thereby activating intracellular drugs and disrupting autophagy. This system achieved a 92.2% tumor inhibition rate and 5.08× improved efficacy compared to hydroxychloroquine alone [98].

6.6. Biomimetic and Homologous Targeting

Biomimetic targeting strategies enhance immune evasion and tumor localization by replicating native cellular interfaces. For instance, nanoparticles coated with natural killer (NK) cell membranes have demonstrated the ability to cross the BBB and target glioblastoma via tight junction modulation, achieving an 85% tumor inhibition rate and deep-tissue imaging through the skull [27]. Similarly, cancer cell membrane–derived liposomes (MCLs) exploit homotypic antigen recognition to prolong tumor retention and support lymph node mapping. These MCLs enabled effective PDT with >65% tumor suppression and showed high PET imaging fidelity (radiochemical purity >98%) without systemic toxicity [28]. Collectively, biomimetic systems offer prolonged circulation, high biocompatibility, and enhanced specificity by mimicking natural cellular functions.

6.7. Dual and Multimodal Targeting Strategies

Dual and multimodal targeting strategies integrate multiple mechanisms to enhance delivery precision and overcome biological barriers. One example includes hyaluronic acid–functionalized nanoparticles conjugated with magnetic Prussian blue, which combine CD44 receptor targeting and magnetic guidance to enhance tumor accumulation and achieve >89.95% tumor inhibition [38]. Similarly, nanoparticles dual-modified with lactoferrin and RGD peptides have shown effective BBB penetration and glioma-specific binding, supported by high T₁ relaxivity (r₁ = 60.8 mM⁻¹·s⁻¹) and a 423 ± 42% increase in signal-to-noise ratio in glioblastoma models [56]. Sequential targeting designs are also emerging; for example, IR825@Bev-PLGA-PFP nanoparticles first bind VEGF receptors to localize in tumor vasculature, followed by NIR-induced mitochondrial activation for photothermal therapy, resulting in complete tumor ablation and multimodal imaging capability [7].

Targeting strategies in nanomedicine exemplify the convergence of engineering, molecular biology, and clinical translation. From ligand-mediated recognition to microenvironmental responsiveness, magnetic manipulation, and biomimicry, each approach contributes unique strengths. The most effective systems integrate multiple mechanisms—enabling precise, programmable delivery and synergistic imaging–therapy functionality. Yet, addressing tumor heterogeneity and dynamic microenvironmental shifts remains essential for maximizing therapeutic efficacy. Continued innovation in this area is vital to advancing next-generation nanotheranostics platforms toward clinical success. Scheme 3 provides a visual summary of major targeting modalities—active, passive, stimuli-responsive, magnetic, organelle-specific, and biomimetic—within a unified framework. As the field advances, personalized biomarker-driven targeting and adaptive delivery systems that dynamically respond to tumor evolution are expected to further improve clinical translation and therapeutic outcomes in nanomedicine.

7. Therapeutic Modalities Enabled by Nanocarriers

Nanomedicine has redefined therapeutic strategies by enabling targeted delivery, controlled release, and multimodal treatment through unified nanoscale platforms. Designed to navigate physiological barriers and incorporate diagnostic features, these systems underpin the theranostic model. This section reviews key therapeutic modalities supported by nanocarriers—spanning chemotherapy, phototherapies, gene silencing, and catalytic therapy—with an emphasis on synergistic and integrative approaches. In particular, growing interest in combination therapies such as chemo–photothermal and gene–immunotherapy has prompted the development of multifunctional nanoplatforms capable of exploiting overlapping mechanisms to enhance efficacy and overcome resistance.

7.1. Chemotherapy: Targeted and Controlled Cytotoxicity

Nanocarriers enhance chemotherapy by improving drug solubility, pharmacokinetics, and tumor specificity, while minimizing systemic toxicity. Stimuli-responsive mechanisms—such as pH-, redox-, or enzyme-sensitivity—allow for precise spatiotemporal control over drug release. For instance, gold–iron oxide (Fe₃O₄@Au) nanocomposites loaded with lipoic acid–curcumin and GSH enabled pH-triggered DOX release and selective uptake in cancer cells with elevated GSH levels. This system achieved potent cytotoxicity (IC₅₀ = 2.69 μg/mL) and demonstrated strong MRI contrast (r₂ = 80.73 mM⁻¹s⁻¹) [29]. Similarly, redox-responsive lanthanide–cyclen–camptothecin nanocomposites facilitated intracellular drug release in the reducing tumor environment, while offering dual NIR/MR imaging capabilities and tumor selectivity [58]. Dendrimer platforms co-loaded with gadolinium and DOX provided sustained drug release, folate receptor targeting, and integrated diagnostic and therapeutic functionality [59]. Enzyme-immobilized mesoporous silica nanoparticles (IBN-4) encapsulating HRP catalyzed the conversion of indole-3-acetic acid into cytotoxic radicals, inducing apoptosis in HT-29 colon cancer cells with high storage stability and potential for repeated use [18]. However, clinical translation of nanocarrier-based chemotherapies has faced challenges including premature drug leakage, inadequate tumor accumulation, and dose-limiting toxicities in off-target tissues.

7.2. PTT: Heat-Induced Tumor Ablation

PTT utilizes nanocarriers capable of converting NIR light into heat, enabling precise tumor ablation with minimal damage to adjacent tissues. A notable example is porphyrin-based organic (PNPD) nanoparticles, which incorporate porphyrin-based NIR-IIa fluorescence and RGD-targeting moieties. These particles successfully penetrated the BBB, provided photoacoustic imaging, and demonstrated therapeutic efficacy in glioblastoma models [12]. Neutral π-radical nanoparticles based on 10H-spiro(acridine-9,9′-fluorene) (SFA-BTM) enabled NIR-II imaging, high-resolution whole-body angiography, and effective photothermal treatment in bone tumor models [10]. In addition, Janus-like Fe₃O₄-Au(shell)-PEG nanostructures integrated MRI (r₂ = 216 mM⁻¹·s⁻¹) with NIR responsiveness, achieving tumor temperatures of 54.6 °C and complete ablation within six days in breast cancer models [19]. Despite these advances, the efficacy of PTT remains limited by poor light penetration and uneven heat distribution, often resulting in suboptimal tumor eradication and unintended thermal damage to surrounding healthy tissues.

7.3. PDT: Light-Triggered Oxidative Stress

PDT relies on photosensitizers that, when activated by specific wavelengths of light, generate reactive oxygen species (ROS) to induce apoptosis in cancer cells. Nanocarriers enhance PDT by improving photosensitizer solubility, tumor specificity, and controlled activation. For example, aggregation-induced emission luminogen (AIEgen) systems such as D2P1–3CBT conjugates respond to tumor-associated cathepsin proteases, triggering intracellular polymerization that enhances fluorescence retention and disrupts cytoskeletal function, thereby improving photodynamic suppression of tumor growth with minimal off-target effects [32]. Redox-responsive platforms such as α-cyclodextrin–disulfide–chlorin e6 (α-CD-ss-Ce6) nanocarriers respond to elevated GSH concentrations in tumors, releasing chlorin e6 at the site of action and improving PDT efficacy and tumor retention [25]. A particularly innovative approach involves the use of multicompartment membrane-derived liposomes (MCLs), constructed by reassembling cancer cell membrane vesicles with Tween-80 micelles. These MCLs are engineered to load the photosensitizer tetrakis(4-carboxyphenyl) porphyrin (TCPP) and are labeled with the radioisotope ⁸⁹Zr via a deferoxamine (Df) chelator, enabling dual functionality for in vivo PET imaging and PDT. The ⁸⁹Zr-Df-MCLs exhibit excellent radiochemical stability, efficient tumor targeting via the enhanced permeability and retention (EPR) effect, prolonged tumor retention, and gradual hepatobiliary clearance. Additionally, they support lymph node mapping and real-time imaging, offering a comprehensive nanotheranostic platform for integrated oncology applications, as shown in Figure 5 [28]. Nonetheless, PDT remains constrained by limited tissue penetration of activating light and hypoxic tumor microenvironments, which reduce ROS generation and therapeutic efficacy.

7.4. Radiotherapy and Radiosensitization: Precision Radiation Enhancement

Nanocarriers augment radiotherapy either by delivering therapeutic radionuclides or by acting as radiosensitizers to intensify radiation effects. A self-assembling nano prodrug (EB–camptothecin, EB-CPT), incorporating Evans blue and radiolabeled with therapeutic ¹⁷⁷Lu or diagnostic ⁶⁴Cu/⁶⁸Ga, enabled single-dose chemo-radio-theranostics. This system demonstrated tumor-specific accumulation, favorable biodistribution, and translational potential, as shown by preclinical studies and a first-in-human diagnostic imaging trial using ⁶⁸Ga-labeled EB-CPT [35].

HSA nanoparticles labeled with ¹²⁵I exhibited enhanced tumor uptake and retention following X-ray exposure, attributed to the X-ray–induced upregulation of caveolin-1, thereby improving SPECT/CT imaging contrast [78]. In a complementary approach, functionalization of HSA nanoparticles with the A15 peptide enabled targeted accumulation in tumor-associated thrombosis induced by irradiation, which enhanced localization of ¹³¹I-labeled HSA for therapeutic purposes in a subsequent study [79].

Furthermore, Gd₂O₃ nanoparticles (ES-GON-rBSA-LF-RGD₂), functionalized with lactoferrin and RGD₂ peptides, penetrated the blood–brain barrier, acted as potent glioblastoma radiosensitizers, and achieved high MRI contrast with a ΔSNR of~423% at 12 h post-injection [56]. However, clinical translation remains hindered by challenges such as non-uniform nanoparticle distribution, off-target radiation amplification, and regulatory complexities.

7.5. Magnetic Hyperthermia: Magnetically Controlled Heat Therapy

Magnetic hyperthermia employs alternating magnetic fields to heat magnetic nanoparticles, inducing localized cytotoxicity or triggering drug release. Folate-functionalized GdIO nanoparticles synthesized with a controllable Curie temperature (~400 K) enabled T₁–T₂ dual-mode MRI while supporting magneto-chemotherapy and selective cytotoxicity in breast cancer models [102]. LSMO (lanthanum strontium manganite oxide)–encapsulated micelles co-loaded with DOX exhibited high drug-loading efficiency (60.45%), induced 89% cancer cell death via chemotherapy, and achieved 80% cell death through magnetic hyperthermia alone, while displaying favorable in vivo biodistribution at therapeutic doses [103]. Yet, achieving uniform thermal distribution and minimizing heat loss to surrounding tissues remain major limitations in clinical hyperthermia applications.

7.6. Gene Therapy and Gene Silencing: Molecular-Level Precision

Gene therapy enabled by nanocarriers allows for the delivery of genetic materials such as siRNA or miRNA inhibitors with improved targeting, intracellular uptake, and stability. One example is a Fe₃O₄/polydopamine nanohybrid incorporating miRNA-21 inhibitors and DOX, designed for cascaded gene silencing, chemotherapy, magnetic targeting, and photothermal therapy. This system used miRNA-21 overexpression in tumors to trigger DOX release and gene silencing, halting therapy once miRNA-21 levels dropped, and achieved significant tumor suppression without systemic toxicity [34]. In another system, Fe₃O₄-MWNT hybrids modified with PEI and PEG were loaded with hTERT-siRNA. These nanoparticles enabled magnetic guidance, MRI visibility, near-infrared-triggered photothermal heating, and efficient gene delivery, supporting combined imaging and gene–photothermal therapy [104]. Nonetheless, gene delivery continues to face significant barriers, including degradation in the bloodstream, immune activation, and inconsistent transfection efficiency across heterogeneous tumor environments.

7.7. Sonodynamic and Catalytic Therapies: Deep-Tissue and Oxidative Strategies

Sonodynamic therapy (SDT) uses ultrasound to activate sonosensitizers, enabling deep-tissue ROS production, while catalytic therapies exploit Fenton or Fenton-like reactions to induce oxidative stress selectively in tumor cells. For example, PHPMR nanoparticles composed of MnFe₂O₄, hematoporphyrin monomethyl ether (HMME), and perfluoropentane (PFP) enabled MRI, ultrasound, and photoacoustic imaging, and supported anti-angiogenic SDT to reduce neo vessel density and stabilize atherosclerotic plaques in vivo [105]. Similarly, CeO₂-Fe₃O₄-chitosan composites co-loaded with methotrexate and Cyanine5 (Cy5) dye provided MRI visibility, cancer-selective ROS modulation, and sustained suppression of tumor growth without post-treatment recurrence [20]. However, limited ultrasound penetration in deep tissues, along with variability in tumor microenvironment pH and redox conditions, can compromise treatment reproducibility and safety.

7.8. Multimodal and Theranostic Platforms: Integrated Therapy and Imaging

Theranostic nanocarriers unify therapeutic and imaging functions, allowing for real-time treatment monitoring and adaptive therapy. For instance, PNDI-2T semiconducting polymer nanoparticles demonstrated high photothermal conversion efficiency (45%) and supported both PTT and PDT, along with PAI for precise tumor mapping and response tracking [31]. Nanographene oxide-PEG (NGO-PEG) nanocarriers co-loaded with paclitaxel (PTX) and indocyanine green provided a triple-action modality—chemotherapy, photothermal therapy, and NIR FI—achieving complete tumor suppression and sustained remission without toxicity over a 30-day period [43]. Iron–platinum (Fe-PRn) nanoparticles, engineered with tunable folate ligand valency, enabled receptor-density–matched drug delivery, MRI-based monitoring, and personalized chemotherapy in heterogeneous tumors [81]. These multimodal systems are particularly suited for combination therapies, where concurrent chemo-photo or gene-immunotherapy approaches can synergistically enhance treatment outcomes and circumvent monotherapy limitations.

Nanocarrier-enabled therapeutic modalities offer unprecedented control in cancer treatment by integrating site-specific localization, controlled drug release, multimodal therapies, and real-time imaging. These platforms support synergistic interventions—from targeted chemotherapy and gene silencing to phototherapy, radio sensitization, and catalytic therapies—tailored to the molecular landscape of individual tumors. Nonetheless, clinical translation remains hindered by pharmacokinetic variability, manufacturing scale-up limitations, and tumor heterogeneity. Bridging the gap between preclinical innovation and clinical application will depend on the continued advancement of integrated, patient-specific theranostic systems that seamlessly unify targeting, diagnostics, and multimodal therapy within a single platform.

8. Imaging and Diagnostic Functions of Nanoplatforms

This section reviews key imaging modalities enabled by nanotechnology and highlights their integration within image-guided therapeutic frameworks.

8.1. FI: High Sensitivity and Molecular Resolution

FI provides high sensitivity, real-time monitoring, and molecular-level resolution, making it a powerful tool for tumor detection and image-guided therapy. Nanoprobes such as carbon dots, QDs, and AIE nanoparticles have improved photostability, signal intensity, and biological compatibility. NIR/NIR-II fluorescent platforms in particular enable deep tissue penetration and reduced background interference. For example, NK@AIEdots—composed of NK cell membranes and pyridal [1,2,3] thiadiazole-based semiconducting polymer (PBPTV), a NIR-II semiconducting polymer—enabled through-skull imaging of glioblastoma and facilitated photothermal tumor suppression [27]. SFA-BTM nanoparticles, which incorporate stable π-radical structures, further supported NIR-II imaging of tumor vasculature and acted as a dual-modality platform for photothermal therapy [10]. Activatable systems such as RhB-S(Se)-CPT (Rhodamine B-based prodrug nanoparticle containing selenium and camptothecin) nanoparticles employed redox-cleavable linkers for camptothecin release, while Rhodamine B fluorescence provided real-time visualization of therapeutic activation [36]. Targeted fluorescence probes, including GLUT-binding CPPG AIE dots (nanoparticles formed from a prodrug amphiphile with aggregation-induced emission properties, designed to specifically target glucose transporters on the surface of cancer cells) [73] and chlorin e6 (Ce6)-conjugated GSH-responsive carriers [25], demonstrated tumor-specific accumulation and enhanced therapeutic efficacy, enabling improved PDT through prolonged retention and localization. However, when integrated with other imaging modalities such as MRI or PAI, fluorescence systems may suffer from signal interference or quenching, often caused by energy transfer between contrast agents. This necessitates careful spectral and spatial separation to preserve imaging fidelity.

8.2. MRI: Deep Tissue Contrast and Functional Guidance

MRI is renowned for its deep tissue penetration, superior spatial resolution, and soft tissue contrast. Nanocarriers incorporating iron oxide (Fe₃O₄) for T₂ contrast or gadolinium (Gd³⁺) for T₁ contrast can significantly improve image clarity and anatomical resolution. A notable example is the cRGD-GdIO-DTX platform, a dual T₁-T₂ MRI system that targeted αvβ3 integrins in pancreatic cancer, enabled acid-triggered docetaxel release, and supported MRI-guided therapy [8]. Folate-modified Gd-loaded dendrimers (G5·NHAc-DOTA(Gd)-PEG-FA/DOX) selectively accumulated in tumor tissues and enabled concurrent sustained drug release and real-time imaging [59]. While not assessed for MRI relaxivity, Fe₃O₄–polydopamine carriers exhibited magnetic responsiveness for targeting, gene silencing via anti-miRNA delivery, and photothermal activation, underscoring their utility in multimodal cancer therapy [34]. Multimodal integrations with MRI can be limited by incompatibilities in probe loading or competition for targeting ligands, which may reduce targeting specificity or imaging fidelity.

8.3. PAI: Optical Contrast with Ultrasound Precision

PAI merges the optical contrast of light absorption with the spatial precision of ultrasound, offering deep-tissue imaging with high molecular sensitivity. Semiconducting polymer nanoparticles like PNDI-2T exhibit strong NIR absorption, enabling PAI-guided photothermal and PDT with minimal off-target toxicity [31]. BODIPY-Gd(III) nano discs, assembled via J-aggregation, demonstrated triple functionality—MRI, PAI, and photothermal therapy—within a single nano system [37]. This system successfully harmonized modality performance by leveraging structural design to spatially organize imaging agents and minimize cross-modality interference. DCF-P nanoparticles, approximately 4 nm in size and based on the IDIC-4F chromophore, successfully combined NIR fluorescence and PAI with a high photothermal conversion efficiency of 80.5%, achieving full tumor eradication in-vivo [11].

8.4. Nuclear Imaging (PET/SPECT) and CT: Whole-Body Quantification and Clinical Translation

Positron emission tomography (PET) and single-photon emission CT (SPECT) are essential nuclear imaging techniques for whole-body quantification of radiotracer biodistribution. These modalities are often integrated with CT, which provides anatomical context through X-ray imaging, although CT itself is not a nuclear modality. EB–camptothecin nanomedicine, radiolabeled with positron-emitting isotopes such as ⁶⁴Cu or ⁶⁸Ga, has demonstrated high-resolution PET imaging, prolonged tumor retention, and potential clinical utility in chemoradiotherapy applications [35]. While therapeutic isotopes like ¹⁷⁷Lu do not support PET imaging due to their beta emissions, they are valuable for radiotherapeutic functions when incorporated into nanomedicines. Human serum albumin (HSA) nanoparticles labeled with ¹²⁵I have been shown in preclinical studies to benefit from X-ray–induced caveolin-1 upregulation, enhancing cellular uptake and SPECT/CT imaging contrast [78]. In related but distinct research, ¹³¹I-labeled HSA nanoparticles have been evaluated primarily for therapeutic applications, though imaging efficacy was not reported in the same context. Furthermore, ⁸⁹Zr-labeled multicompartment liposomes derived from tumor cell membranes have enabled PET imaging, lymph node mapping, and PDT, highlighting the multifunctionality of nanotheranostic platforms [28].

8.5. Multimodal Imaging: Combining Modalities for Diagnostic Synergy

Multimodal nanoplatforms integrate two or more imaging modalities—such as FI, MRI, PAI, or CT—to improve diagnostic accuracy, therapeutic guidance, and treatment monitoring. For example, HA–Prussian blue@QD nanoparticles enabled dual MRI and NIR FI, targeted CD44 receptors, and mediated photothermal therapy with 89% tumor inhibition in vivo [38]. In this system, quenching of QD fluorescence by magnetic components was minimized through surface passivation strategies and core–shell engineering. The IR825@Bev-PLGA-PFP system combined fluorescence, photoacoustic, and ultrasound imaging with mitochondria-targeted photothermal therapy in thyroid cancer models [7]. Similarly, pH-responsive polymer-coated multifunctional magnetic (PHPMR) nanoparticles comprising MnFe₂O₄, hematoporphyrin monomethyl ether (HMME), and perfluoropentane (PFP) enabled tri-modal imaging [MRI, photoacoustic, ultrasound] and targeted neovascularization in atherosclerotic plaques, leading to reduced vessel density and improved plaque stability [105]. Although integrating multiple imaging elements can introduce formulation complexity and potential signal interference, strategic material design—such as compartmentalization or orthogonal activation mechanisms—can preserve signal integrity across modalities.

8.6. Stimuli-Responsive Imaging: Smart Activation in Tumor Microenvironments

Stimuli-responsive nanoplatforms are engineered to activate imaging contrast or therapeutic payloads in response to tumor-specific conditions such as pH, redox potential, or aberrant gene expression. Zinc(II) phthalocyanine tetrakis(azaphenyl) [ZnPc(TAP)₄], for example, is a photosensitizer that remains photoinactive at physiological pH but becomes phototoxic under acidic tumor conditions, thereby enabling selective PDT and tumor imaging [33]. QD-HA-PEI nanoparticles were used to deliver anti-miR-27a and provided dual-fluorescent, pH-responsive gene release, along with in-vivo fluorescence tracking and gene silencing efficacy [69]. Although Fe₃O₄/polydopamine–DOX-DNA constructs are not fluorescently activatable by miR-21, they facilitated magnetic targeting, photothermal therapy, and miRNA-triggered chemotherapeutic release, serving as multifunctional platforms [34].

Nanotechnology has fundamentally expanded the diagnostic landscape through multifunctional, responsive imaging platforms. Modalities such as FI, MRI, PAI, nuclear imaging (PET/SPECT), and CT have been successfully integrated into therapeutic systems to support real-time tracking, adaptive treatment, and precision-guided intervention. From activatable probes [33,36], to multimodal constructs [7,37], and clinical candidates [28,35], imaging-enabled nanotheranostics represent the forefront of personalized medicine by offering real-time visualization, adaptive treatment guidance, and molecular-level precision.

Table 3. Integrated imaging and therapeutic strategies in nanoplatforms: theranostic applications.

Imaging-Therapy Integration	Design Features and Representative Nanoplatforms	Therapeutic Applications and Translational Relevance	Supporting Literature
Chemotherapy + Imaging	DOX, PTX paired with MRI, FL using pH-sensitive or receptor-targeted nanocarriers.	Foundational for drug delivery tracking and image-guided dosing	[2,17,23,59,88]
PTT + Imaging	NIR/NIR-II laser-triggered ablation with MRI, FL, or PAI using biocompatible nanoplatforms.	Used in non-invasive thermal therapy and real-time image-guided monitoring.	[3,12,31,106]
PDT + Imaging	Photosensitizers (AIEgen, porphyrins, Ce6) activated by light, paired with PET or FL for theranostics.	Enables ROS-mediated tumor ablation with real-time imaging of activation or localization.	[22,28,71,107]
Chemo + PTT/PDT + Imaging	DOX-based systems integrating NIR-triggered PTT or PDT with FL or optical imaging for combination therapy.	Enables synergistic cancer therapy and real-time image-guided delivery with reduced resistance.	[89,108]
Gene Therapy + Imaging	siRNA/miRNA or DNAzyme-loaded nanoplatforms integrated with FL or MRI for image-guided gene silencing.	Enables targeted genetic modulation and fluorescence/MRI-tracked therapeutic monitoring.	[69,90,104]
Magneto-Therapy + Imaging	Magnetic nanoplatforms (e.g., SPIONs, GdIO, MNP hybrids) enabling MRI-guided hyperthermia or chemotherapeutic delivery.	Useful in deep tumor targeting, magnetically triggered therapy, and non-contact thermal ablation with MRI feedback.	[15,26,50,75,102]
Radiotherapy + Imaging	Nanoplatforms radiolabeled with I-131, Lu-177, or Y-90 for combined radionuclide therapy and PET/SPECT imaging, enabling real-time dosimetry and tumor tracking.	Enables precise systemic radiotherapy with companion diagnostics and quantitative biodistribution.	[35,39,109,110,111]
FL Imaging + Multiple Therapies	Nanoplatforms incorporating visible/NIR/NIR-II fluorescence for subcellular tracking of drug release, photothermal/photodynamic response, or gene delivery.	Enables preclinical real-time therapy-response monitoring, improving therapeutic scheduling and tumor specificity.	[1,9,25,46,74,112]
MRI + multi-Therapies	T1/T2 MRI-guided nanoplatforms combining chemotherapy or photothermal therapy, often via pH-sensitive or ligand-targeted drug release.	Enables real-time, clinical-grade tumor visualization, drug tracking, and therapy response monitoring.	[2,8,17,19,55,81]
PAI + PTT/PDT/Drug Tracking	Photoacoustic contrast agents integrated with NIR-triggered photothermal or photodynamic therapies, often using semiconducting polymers or dye-loaded carriers.	Enables real-time, deep-tissue imaging of thermal or oxidative stress with simultaneous tumor ablation or drug response monitoring.	[11,37,66,70,113]
PET/SPECT/CT + Therapy	Nanocarriers labeled with PET/SPECT/CT isotopes co-delivering chemotherapeutic, PDT, or radiotherapeutic agents.	Enables full-body biodistribution tracking, dose planning, and image-guided therapy in preclinical or clinical theranostic frameworks.	[28,35,64,78,110]
Multimodal Imaging + Therapy	Hybrid nanoplatforms integrating two or more imaging modalities (e.g., MRI/FL, MRI/PAI, US/FL) with PTT, chemotherapy, or PDT.	Supports pre-treatment planning, real-time intra-treatment monitoring, and post-treatment evaluation in preclinical models.	[7,23,37,38,112]
pH-Responsive Therapy + Imaging	Smart nanocarriers triggered by tumor acidity for controlled drug release or imaging contrast activation, often using MRI or fluorescence.	Enables tumor-specific drug release and imaging, minimizing systemic toxicity in preclinical models.	[17,24,29,51,88]
Redox-Responsive Therapy + Imaging	Nanoparticles that activate drug release or imaging signals via tumor-associated GSH or ROS levels, using disulfide linkers, ROS-sensitive structures, or fluorescence switching.	Enables selective drug delivery and imaging in reductive or oxidative tumor environments, improving specificity and reducing systemic effects.	[36,46,54,58,74]
Light-Activated Theranostics	NIR-triggered (808–1064 nm) nanoplatforms integrating PTT or imaging agents (NIR-II FL, PAI) with tumor-targeting elements (e.g., BBB-crossing, cell membrane camouflage, charge switching).	Enables high-resolution imaging and spatially confined therapy, suitable for brain tumors, surgical guidance, or minimally invasive treatment.	[12,27,106,114,115]
Ultrasound-Triggered Theranostics	Acoustic droplet vaporization or LIFU-triggered drug release using nanobubbles or polymer nanocarriers with ultrasound/MRI/photoacoustic imaging.	Enables non-invasive, real-time therapy in deep tissues (e.g., kidney, vascular plaques, brain); useful in oncology and organ-specific disease.	[48,85,105]
Ligand-Targeted Systems + Imaging	Nanoparticles functionalized with ligands (e.g., RGD, folate, HA, octreotide) for receptor-mediated targeting and visualized via MRI or fluorescence.	Improves therapeutic index and imaging precision; valuable for receptor-overexpressing tumors and personalized cancer therapy.	[8,23,59,69,116,117]

summarizes the primary classes of imaging-therapy integration, highlighting design features, nanoplatforms, and their therapeutic applications.

9. In Vitro Evaluation and Biocompatibility

Biocompatibility and in vitro evaluation are essential for establishing the safety and efficacy of nanoparticle-based biomedical systems. These assessments are particularly critical for identifying platforms that exhibit selective cytotoxicity against cancer cells while preserving normal tissue viability. As nanomedicine advances toward clinical application, robust in-vitro characterization provides a foundational filter for translational success. However, in-vitro models often fail to capture complex immune responses such as complement activation, opsonization, and clearance by mononuclear phagocyte systems that can significantly affect nanoparticle fate in-vivo.

9.1. Assessing Biocompatibility: Standard Assays and Normative Models

Biocompatibility is commonly evaluated through standard cytotoxicity assays such as MTT, LDH leakage, hemolysis, and general cell viability assays conducted across both cancerous and non-cancerous cell lines. A recurring observation in these studies is that nanomaterials in their imaging-only or inactive form typically display negligible cytotoxicity, while therapeutic effects are observed only under condition-specific activation.

For example, molybdenum disulfide-gadolinium-bovine serum albumin (MoS₂-Gd-BSA) nanocomposites synthesized via biomineralization and amide coupling showed minimal cytotoxicity under physiological conditions. However, under 808 nm laser irradiation, these nanocomposites achieved effective photothermal tumor ablation and dual MR/PA imaging in tumor models [3]. PEGylated ferrite nanoparticles also demonstrated high biocompatibility at therapeutic concentrations. PEGylation improved dispersion, reduced aggregation, and minimized toxicity, while maintaining imaging contrast and magnetic hyperthermia capabilities [21]. Similarly, mesoporous silica nanocarriers loaded with HRP were non-toxic under normal conditions but triggered apoptosis in cancer cells when indole-3-acetic acid was enzymatically converted to cytotoxic radicals, as confirmed through in-vitro assays [18]. In another example, CQDs derived from almond resin using a green synthesis method exhibited excellent fluorescence (quantum yield: 61%) and showed no detectable cytotoxic effects, supporting their application in high-resolution, multicolor cellular imaging [42]. Nonetheless, standard cytotoxicity assays may overlook nanoparticle-induced immune recognition pathways—such as Toll-like receptor (TLR) signaling or NLRP3 inflammasome activation—that contribute to inflammation and immune clearance in-vivo.

9.2. Selective Cytotoxicity and Tumor-Specific Action

Selectivity in cytotoxicity is a critical requirement for nanomedicine platforms to reduce damage to healthy tissues. Ligand-mediated targeting and tumor microenvironment–specific responsiveness are key mechanisms to achieve this goal. FA/GODs-Gd(DTPA)/DOX nanoparticles, for example, demonstrated strong cytotoxicity against H460 lung cancer cells while sparing healthy HLF cells, confirming their tumor specificity [23]. Likewise, AS1411 aptamer-dendrimer-Iohexol conjugates selectively targeted nucleolin-overexpressing MCF-7 breast cancer cells and showed negligible toxicity toward normal cells [13]. Gold/polymer nanoconjugates activated fluorescence and induced selective cytotoxicity in MCF-7 cells in response to intracellular GSH and acidic pH, with minimal effect on non-targeted HEK-293T cells [74]. Furthermore, zinc phthalocyanine photosensitizers with tetraazaporphyrin side groups [ZnPc(TAP)₄] exhibited potent phototoxicity under acidic tumor conditions (IC₅₀ = 0.20 μM), while maintaining minimal toxicity at physiological pH, underscoring their environmental selectivity [33]. However, tumor-selective effects observed in vitro may be attenuated in-vivo due to non-specific uptake by macrophages or dendritic cells following opsonization and chronic exposure.

9.3. Confirming Safety in Normal Cells and In-Vivo Models

Ensuring that nanocarriers do not harm healthy tissues is vital for clinical translation. This requires extended safety evaluations, not only in vitro using normal cell lines, but also in in vivo animal models. GLUT-targeted CPPG AIE dots, for instance, selectively inhibited tumor cell proliferation while exhibiting negligible toxicity in non-cancerous cells [73]. Multifunctional nanoprobes composed of gold nanorods, mesoporous silica, gadolinium, folate, and PEG enabled effective tumor ablation in murine models. These nanoprobes demonstrated low systemic toxicity and favorable metabolic clearance, supporting their clinical translational potential [40]. Importantly, in-vivo testing captures immune responses absent in-vitro, including splenic accumulation, complement-mediated opsonization, and cytokine induction, all of which can influence circulation half-life and biodistribution.

9.4. Visualization-Integrated Viability Monitoring

Many advanced nanoplatforms integrate imaging functions with therapeutic effects, enabling dynamic, real-time tracking of treatment efficacy and biodistribution. For example, cRGD-GdIO-DTX nanoclusters provided both T₁ and T₂ MRI contrast alongside pH-sensitive docetaxel release, enabling effective tumor visualization and targeted therapy in pancreatic cancer models [8]. Quantum dot–functionalized polymeric nanoparticles have also been used for multimodal imaging and to overcome drug resistance in cancer treatment strategies [118]. Fe₃O₄/polydopamine hybrids activated by miRNA-21 enabled magnetic targeting, photothermal therapy, and gene silencing, demonstrating strong tumor specificity and systemic safety [34]. Activatable imaging systems such as ZnPc(TAP)₄ photosensitizers [33] and GLUT-targeted CPPG AIE probes [73] successfully combined cytotoxic effects with imaging to provide comprehensive preclinical validation. These systems offer valuable feedback in-vitro yet may require further optimization to evade immune detection and chronic accumulation during in-vivo deployment.

In-vitro evaluation of nanoplatforms integrates multiple critical factors: cytotoxicity profiling, biocompatibility testing, stimuli-responsiveness, and imaging feedback. Together, these components ensure therapeutic precision and systemic safety. Platforms like miRNA-responsive hybrids [34], pH-activated photosensitizers [33], and GLUT-targeted visualizable probes [73] exemplify how nanomedicine is converging safety and selectivity for clinical translation. Nevertheless, bridging the gap between in vitro immune tolerance and in vivo immunogenicity remains a key challenge, particularly in addressing long-term immune surveillance, complement activation, and off-target clearance mechanisms. These preclinical studies serve as indispensable checkpoints for identifying nanotheranostics systems that can deliver tumor-specific treatments while minimizing off-target toxicity.

10. In-Vivo Performance and Biodistribution

Effective clinical translation of nanoparticle-based therapies hinges on their ability to achieve precise tumor targeting, favorable biodistribution, sustained therapeutic efficacy, and systemic safety. In vivo studies serve as critical preclinical evaluations to assess these attributes, including selective tumor accumulation, therapeutic response, clearance mechanisms, and integrated imaging capabilities. The emergence of multifunctional theranostic platforms continues to enhance the precision and personalization of nanomedicine. However, immune clearance and recognition by the mononuclear phagocyte system (MPS), particularly in the liver and spleen, remain significant barriers to prolonged circulation and effective tumor delivery.

10.1. Tumor-Selective Accumulation and Retention

Selective accumulation and retention of nanocarriers at tumor sites is a central goal of nanomedicine, achieved through passive and active targeting mechanisms. Passive targeting leverages the EPR effect, while active targeting employs ligands such as RGD peptides, folic acid, or monoclonal antibodies to bind specific receptors overexpressed on tumor cells. For instance, cRGD-GdIO-DTX nanoclusters exhibited robust tumor accumulation in pancreatic cancer models by targeting αvβ3 integrins. This led to enhanced MRI contrast and pH-triggered docetaxel release, providing a dual therapeutic and diagnostic benefit [8]. Similarly, CD44-targeted magnetic Prussian blue nanoparticles achieved over 89% tumor growth inhibition in-vivo following NIR irradiation and magnetic field–mediated enrichment, confirming precise tumor localization and effective photothermal therapy [38]. PEGylated nano-GO (NGO-PEG) co-loaded with paclitaxel and ICG also demonstrated prolonged retention within tumors, with complete tumor suppression and no relapse observed over 30 days in treated mice, all while exhibiting minimal systemic toxicity [43]. Despite these advances, deep tissue penetration remains limited by factors such as high interstitial fluid pressure and dense extracellular matrix, which can hinder uniform nanoparticle distribution within solid tumors.

10.2. Multimodal Imaging for Real-Time Biodistribution and Therapy Guidance

Multimodal imaging enables real-time, non-invasive monitoring of nanoparticle distribution, activation, and therapeutic response, enhancing precision and adaptability during treatment. Gd₂O₃/CuS nanodots, for example, supported both FI and MRI, enabling dual-mode tumor localization and photothermal guidance [49]. PNPD nanoparticles, composed of porphyrin nanostructures, provided high-resolution NIR-IIa imaging of glioblastoma, with enhanced BBB penetration, high signal-to-noise ratios, and low systemic toxicity [12]. Furthermore, ⁸⁹Zr-labeled tumor membrane–derived liposomes facilitated PET imaging and lymph node mapping, with extended in-vivo retention and no detectable systemic toxicity, highlighting their translational potential [28]. Nonetheless, rapid uptake by the reticuloendothelial system (RES), especially in hepatic and splenic tissues, can reduce tumor accumulation and compromise therapeutic efficacy, emphasizing the need for stealth coatings and surface modifications.

10.3. In-Vivo Therapeutic Efficacy: Tumor Suppression and Ablation

In-vivo therapeutic efficacy is typically assessed using xenograft or orthotopic tumor models to evaluate the capacity of nanocarriers to induce significant tumor suppression or complete ablation. These effects are particularly evident in multifunctional platforms that combine imaging with thermal or photodynamic therapies. For instance, Fe₃O₄-Au Janus nanohybrids have demonstrated strong photothermal performance under NIR irradiation. In treated mice, intratumoral temperatures reached 54.6 °C within 5 min of 0.65 W/cm² laser exposure, surpassing the therapeutic threshold for irreversible tumor cell damage. In contrast, the control group receiving PBS and laser showed a maximum tumor temperature of only 43.7 °C, insufficient for effective ablation. As shown in Figure 6, the Fe₃O₄-Aushell-PEG + laser group exhibited rapid tumor regression, with relative tumor volume decreasing from 1.0 to 0.55 by day 2 and further to 0.24 by day 3. By day 6, no visible tumor remained—only a residual black scar—while tumors in the untreated, PBS+laser, and Fe₃O₄-Aushell-PEG (no laser) groups continued to grow, reaching relative volumes of 3.63–3.85 by day 8 and exceeding 5.0 by day 14. Histological evaluation (Figure 6E–G) revealed viable morphology in control group tumor tissues, with no signs of necrosis or apoptosis, underscoring the importance of photothermal activation. Importantly, the biosafety of the Fe₃O₄-Aushell-PEG nanoplatform was also confirmed. Mice maintained stable body weight (Figure 6A) and showed no behavioral abnormalities throughout the study. Histological examination of major organs (Figure 6I) at day-14 post-treatment showed no observable signs of inflammation or toxicity, indicating excellent systemic compatibility [19]. Still, long-term toxicity and potential nanoparticle accumulation in non-target organs should be systematically evaluated through extended follow-up studies to assess chronic effects.

In addition to photothermal systems, other nanocarriers such as ZnPc(TAP)₄ photosensitizers have demonstrated robust PDT in acidic tumor microenvironments, selectively destroying cancer cells while sparing healthy tissue [35]. Similarly, magnetothermal composites like magnetic nanoparticle @conjugated poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (MNP@PEDOT:PSS) have enabled MRI-guided hyperthermia for image-controlled tumor ablation, further illustrating the power of theranostic platforms to integrate precision imaging with targeted therapy [39].

10.4. Biosafety and Systemic Clearance

Ensuring systemic biosafety and favorable clearance profiles is vital for any nanoplatform intended for clinical use. These properties are typically validated through histological analyses, blood chemistry panels, and organ function studies in animal models. Many nanocarriers demonstrate renal or hepatobiliary clearance pathways, coupled with low off-target toxicity and good tolerability. For example, ¹⁷⁷Lu-labeled EB-CPT nanoconjugates showed effective tumor suppression and low systemic toxicity in preclinical models and also supported favorable biodistribution and safety in early-phase human studies [85]. Likewise, ¹²⁵I-HSP-PEG nanoparticles exhibited efficient tumor uptake, dual renal and fecal clearance, and no signs of organ damage across both xenograft and patient-derived xenograft (PDX) models [81]. Notably, in a non-oncologic application, TRPC6-targeted dexamethasone-loaded nanobubbles reduced fibrosis and inflammation at half the standard dexamethasone dose, demonstrating excellent safety with no systemic side effects, indicating potential applications beyond oncology [51]. Nonetheless, nanoparticle accumulation in macrophage-rich organs such as the liver, spleen, and lungs poses a risk for chronic toxicity, necessitating long-term surveillance and comprehensive immunotoxicity assessments.

10.5. Advanced Targeting Strategies and Personalized Delivery

Advanced nanoplatforms are being engineered to support personalized medicine through receptor-density–matched delivery and adaptive targeting strategies. For example, Fe-PR4 and Fe-PR8 (iron–platinum nanostructures functionalized with folate ligands of varying valency) exhibited differential accumulation in tumors based on folate receptor expression levels. Their therapeutic efficacy correlated directly with receptor density, demonstrating potential for receptor-level personalization of cancer treatment [8]. In another study, anti-VEGF–conjugated nanoparticles enhanced MRI contrast while selectively targeting Hep G2 liver cancer cells. This receptor-specific approach enabled guided delivery and improved therapeutic accuracy [28].

Such precision-targeting strategies represent a promising direction in patient-specific nanomedicine, particularly in heterogeneous tumor environments where conventional treatments may fall short. As these technologies advance into clinical trials, future research should continue to focus on optimizing biodistribution, enhancing systemic clearance, and refining delivery mechanisms based on individualized tumor biology. Addressing physiological barriers to nanoparticle delivery, such as immune recognition, extracellular matrix density, and endothelial permeability, will be essential for improving intratumoral distribution and clinical outcomes. Continued innovation will accelerate the translation of multifunctional nanomedicine platforms—such as cRGD-GdIO-DTX clusters [35], ⁸⁹Zr-labeled tumor membrane liposomes [28], and radiolabeled EB–CPT nanoconjugates [35]—into next-generation image-guided cancer therapies that achieve high efficacy with minimal systemic burden.

Table 4. Strategic Trends in Tumor-Targeted Nanomedicine: In vivo Selectivity, Efficacy, and Safety.

Theranostic Design Focus	Representative Approaches	Implications for Design and Translation	Supporting Literature
Tumor-Specific Targeting and Selective Cytotoxicity	Receptor-mediated uptake or tumor microenvironment (TME)-responsive activation to preferentially accumulate in tumors and minimize off-target effects.	Emphasize tumor-specific delivery to enhance therapeutic efficacy and reduce systemic toxicity in solid tumors.	[1,8,33,38,68,69,84,119]
Ligand/Peptide/Antibody-Mediated Targeting	Functionalization with ligands such as folate, transferrin, RGD, CD44 antibodies to increase tumor selectivity and receptor-specific uptake.	Match ligand design to receptor expression profiles for patient-specific and tumor-type-specific targeting strategies.	[8,38,57,59,69,93,96,117]
Stimuli-Responsive Drug Release	Triggered therapeutic release based on tumor-specific cues (e.g., acidic pH, high GSH, ROS, NIR light) to achieve spatiotemporal control.	Engineer adaptable nanocarriers to match intratumoral heterogeneity and optimize on-site activation.	[9,24,25,36,83,108]
Biocompatibility and Low Systemic Toxicity	Evaluated through biodistribution studies, histology, blood chemistry, and weight monitoring; confirmed safety in vitro and in vivo across nanoplatforms.	Incorporate biosafety screening early in development to meet preclinical safety and regulatory standards.	[3,11,28,39,43,86]
Imaging-Guided Theranostics	Multimodal platforms (MRI, PET, PA, NIRF, SWIR) enable tumor visualization, real-time monitoring, and guided therapy with high spatial and temporal resolution.	Embed imaging functionalities during nanoparticle design to facilitate precision therapy and noninvasive tracking.	[4,11,28,49,56,112,120]
Photothermal and Photodynamic Therapy (PTT/PDT)	Light-triggered nanotherapeutics utilizing NIR/NIR-II wavelengths (e.g., 1064 nm) for tumor ablation via thermal (PTT) or oxidative (PDT) mechanisms. Some systems combine both modalities for synergistic effects.	Focus on clinically relevant NIR-II wavelengths to improve tissue penetration, treatment depth, and translational applicability.	[9,11,41,77,106,114,121]
Multimodal or Synergistic Therapy	Co-delivery of chemo, gene therapy, PTT, PDT, or radiotherapy in programmable, stimulus-responsive platforms for enhanced tumor killing and reduced resistance.	Optimize combinatorial regimens, treatment sequencing, and nanocarrier design to maximize synergy and therapeutic outcome.	[34,35,95,99,122]
BBB Penetration/CNS Targeting	BBB-crossing ligands (RGD, Angiopep-2, lactoferrin) and biomimetic membranes (e.g., NK cells) enable delivery across the BBB and accumulation in gliomas.	Confirm therapeutic efficacy and imaging performance in orthotopic glioblastoma models to validate CNS-targeted nanoplatforms.	[12,27,56,123]
Real-World & Translational Models	Use of orthotopic, patient-derived xenografts (PDX), and first-in-human data to evaluate nanotheranostics in clinically relevant settings.	Bridge preclinical and clinical gaps by incorporating advanced in vivo models and early-phase translational studies.	[35,39,65,99]
Disease Applications Beyond Cancer	Adaptation of nanoplatforms for non-cancer conditions including nephropathy, atherosclerosis, and lymphatic diseases; platforms provide imaging and targeted therapy.	Expand theranostic applications to inflammatory and chronic metabolic diseases by leveraging versatile nanocarrier designs.	[85,105]

summarizes the primary classes of theranostic design, highlighting representative approaches, and their implications for design and translation.

11. Challenges and Translational Strategies in Nanotheranostics

Despite transformative progress, nanotheranostic systems face several critical barriers to clinical translation. The very features that make these platforms powerful—such as multifunctionality, stimuli-responsiveness, and integrated imaging—also introduce significant challenges related to design complexity, reproducibility, biosafety, tumor penetration, and regulatory compliance. These issues restrict scalability, undermine long-term safety evaluations, and create obstacles to achieving translational fidelity. Clinical evidence highlights both the potential and pitfalls of these systems. For example, BIND-014, a prostate-specific membrane antigen (PSMA)-targeted nanoparticle carrying docetaxel, demonstrated promising anti-tumor activity, PSA responses, and circulating tumor cell conversions in chemotherapy-naïve metastatic castration-resistant prostate cancer patients, while maintaining a manageable toxicity profile in Phase II trials [124]. Earlier Phase I data further confirmed its tolerability, pharmacokinetic advantages, and antitumor effects across multiple solid tumors, including those without detectable PSMA expression, reflecting both efficacy and the need for broader targeting strategies [125]. Likewise, CYT-6091, a gold nanoparticle conjugated with tumor necrosis factor-alpha (TNF-α), enabled systemic delivery of TNF-α at doses previously considered too toxic, with nanoparticles selectively localizing in tumor tissue, demonstrating the feasibility of nanocarrier-enhanced delivery while underscoring the need for careful immune and safety modulation [126]. These examples underline the dual nature of nanotheranostics: while offering precise delivery and therapeutic enhancement, they pose persistent translational hurdles tied to pharmacokinetics, heterogeneity in tumor uptake, and regulatory alignment. Overcoming these limitations through standardized production, improved predictive models, and integrated regulatory frameworks is essential for advancing nanotheranostics toward routine clinical application.

11.1. Current Limitations

Complexity and manufacturing challenges: Many nanotheranostics systems feature sophisticated architectures—including hybrid cores, multilayered shells, and multi-ligand surface modifications—that are highly effective for multifunctional delivery and imaging. However, these complex designs present significant challenges in terms of reproducibility, batch-to-batch uniformity, and scalability under GMP standards. GMP-compliant production often requires advanced instrumentation, extensive purification steps, and tight process control, all of which limit throughput and increase cost. For instance, Janus Fe₃O₄–Au nanohybrids and Gd₂O₃/CuS nanocomposites exemplify engineering excellence but are difficult to synthesize consistently at scale, thus impeding their transition to clinical-grade production [19,49]. Furthermore, real-world deployment requires long-term stability; many formulations suffer from limited shelf life due to aggregation or degradation under storage conditions.

Tumor heterogeneity and limited penetration: Tumor heterogeneity remains a formidable barrier to uniform intratumoral delivery. Even actively targeted platforms that employ folic acid ligands [2] or cRGD peptides for integrin recognition [8] often exhibit uneven drug distribution within tumor tissues. This issue arises due to variable receptor expression, high interstitial fluid pressure, and dense extracellular matrices that prevent deep nanoparticle penetration. Although nanocarriers such as NGO-PEG co-loaded with ICG and paclitaxel show promising tumor retention, they still struggle to achieve homogeneous penetration throughout solid tumor masses [43]. In addition, poor endosomal escape further reduces cytosolic drug availability, especially for nucleic acid-based or enzymatically activated therapeutics.

Long-term biosafety concerns: While many inorganic nanomaterials demonstrate short-term biocompatibility, their long-term biological fate remains incompletely understood. Accumulation of materials such as gold, iron oxide, or QDs in organs over extended periods may provoke chronic inflammation, interfere with normal tissue function, or generate toxic degradation byproducts. For instance, quantum dot–polymer constructs and CuS-based nanodots raise concerns regarding persistent tissue retention and long-term safety [49,118]. Despite favorable acute toxicity profiles reported in clearance studies with PEG-based and iodinated constructs [21,39], these platforms still require prolonged observation to ensure safe degradation and elimination. Chronic accumulation, especially in the liver and spleen, poses additional risks due to limited metabolic clearance and uncertain biodegradation pathways.

In-vivo stimuli variability: Stimuli-responsive nanoplatforms often exhibit exceptional functionality under control in vitro conditions but can be compromised by physiological variability in-vivo. Tumor-specific stimuli such as pH, redox potential, or enzyme activity may vary considerably between patients, tumor types, and even within different regions of the same tumor. For instance, the ZnPc(TAP)₄ photosensitizer shows strong pH-dependent phototoxicity under acidic conditions [33], but inconsistencies in tumor acidity can result in reduced activation, attenuated therapy, and unpredictable outcomes in vivo. Moreover, systemic conditions such as inflammation or off-target enzymatic activity may trigger premature activation, undermining tumor specificity and increasing off-target toxicity.

Preclinical model inadequacies: Standard preclinical models, particularly subcutaneous xenografts in immunodeficient mice, lack the complexity of human tumors, including immune interactions, pharmacokinetics, and intratumoral heterogeneity. Although such models have enabled foundational work with systems like cRGD-GdIO-DTX clusters [8] and PNPD NIR-IIa imaging nanoparticles [12], they fall short of replicating the full clinical landscape. Even advanced platforms like 177Lu-EB–CPT conjugates—now in first-in-human trials—face added regulatory scrutiny due to their integrated diagnostic and therapeutic nature [35]. The lack of standardized evaluation protocols across labs also complicates comparative assessment of safety, efficacy, and pharmacokinetics.

11.2. Strategic Pathways Forward

To bridge the gap between laboratory innovation and clinical implementation, future nanotheranostics systems must focus on modularity, biosafety, translational predictability, and personalized adaptability. Strategic redesigns should simplify synthesis, support patient-specific deployment, and reduce long-term toxicity risk.

Modular, Scalable Designs: Next-generation nanoplatforms should adopt modular configurations where targeting, imaging, and therapeutic functionalities are physically or chemically decoupled but remain integrable. This allows flexible optimization and streamlined manufacturing. Fe₃O₄@PDA@anti-miRNA/DNA hybrids are a representative example, enabling miRNA-responsive gene silencing, magnetic targeting, and photothermal therapy within a modular framework that supports scalable synthesis [34]. Such modularity enhances reproducibility, facilitates regulatory review, and allows platform customization for different clinical indications.

Biomarker-Guided Personalization: Stratified treatment strategies based on individual tumor biology can enhance treatment efficacy and minimize off-target effects. Personalized delivery guided by molecular biomarkers—such as folate receptor density or gene expression profiles—can be achieved using receptor-responsive systems. For instance, Fe-PR4 and Fe-PR8 nanoparticles demonstrated differential tumor accumulation and efficacy directly correlated with folate receptor density, highlighting the potential for personalized, biomarker-matched therapy [81]. Precision-guided nanomedicine holds particular promise in heterogeneous tumor environments, where molecular profiling can inform platform selection and dosing strategies.

Biodegradable and Organic Nanoplatforms: Shifting toward biodegradable or wholly organic nanomaterials addresses long-term biosafety concerns. Semiconducting polymer systems [31], carbon QDs derived from natural sources [42], and PEGylated platforms [21] exhibit favorable clearance profiles and reduced toxicity. These soft materials support biological metabolism and mitigate risks associated with chronic accumulation and degradation byproducts. Designing carriers with tunable degradation rates also helps synchronize therapeutic release with clearance pathways, minimizing systemic exposure.

Theranostics with Real-Time Imaging Feedback: Theranostic systems that incorporate adaptive imaging—such as MRI, PAI, and fluorescence—allow real-time monitoring of biodistribution, therapy response, and treatment planning. BODIPY–Gd(III) nano discs, for instance, offer integrated MRI and PAI functionalities that guide photothermal therapy with diagnostic precision [37]. Similarly, dual-mode HA–Prussian blue@QD constructs facilitate NIR fluorescence and MRI monitoring while delivering targeted therapy [38]. Emerging closed-loop systems that dynamically adjust drug release based on imaging signals represent a frontier in self-regulating nanomedicine.

PDX and Organoid-Based Validation: To improve the translational relevance of preclinical testing, advanced biological models such as PDX and 3D tumor organoids are increasingly being employed. These systems more accurately reflect human tumor architecture, immune interactions, and drug response. For example, ¹²⁵I-HSP-PEG nanoparticles have been evaluated in PDX models, confirming favorable biodistribution, efficient clearance, and minimal toxicity—outcomes more reflective of clinical behavior [39]. Incorporating these models early in the development pipeline can improve clinical predictability and reduce late-stage attrition.

The clinical advancement of nanotheranostics demands a balance between engineering sophistication and translational feasibility. Future systems must prioritize design simplification without sacrificing functionality, emphasize biocompatible and degradable materials, incorporate dynamic imaging feedback, and validate performance in human-relevant models. Persistent challenges—including design complexity [19,49], tumor penetration limitations [2,43], unresolved biosafety issues [39,118], and physiological variability in responsiveness [33]—must be addressed with translationally aligned, patient-centered strategies. Practical steps such as protocol standardization, robust in-vivo pharmacology testing, and early regulatory engagement will be essential to achieving clinical impact. Only then can nanotheranostics realize their full potential in delivering precision, image-guided, and minimally invasive cancer therapies.

Scheme 4 Illustrates current challenges (e.g., tumor access, biodegradation, regulatory hurdles) alongside future strategies (e.g., modular systems, biomarker-driven therapy, adaptive diagnostics, predictive modeling), offering a roadmap for precision-guided nanomedicine.

12. Conclusions

Nanotheranostic platforms represent a transformative advancement in precision medicine, integrating diagnostic and therapeutic functionalities within a single nanoscale system. This review has detailed the strategic design principles that underpin these systems—including core material selection, surface functionalization, targeting strategies, imaging integration, and stimuli-responsive drug release.

While substantial progress has been made, clinical translation remains hindered by challenges such as architectural complexity, heterogeneous intratumoral penetration, incomplete long-term clearance data, and inter-patient biological variability. Overcoming these barriers will require a shift toward modular, biodegradable, and patient-tailored systems, supported by real-time adaptive imaging and predictive, human-relevant preclinical models.

As nanotheranostics transitions from proof-of-concept to practical application, its future will depend not only on continued materials and bioengineering innovation but also on alignment with scalable synthesis, regulatory approval pathways, and biomarker-driven treatment frameworks. Bridging the gap between laboratory precision and clinical applicability is now the defining challenge—and opportunity—for the next generation of nanomedicine.