Carrier-Free Nanodrugs Reversing Cancer Multidrug Resistance: From Mechanisms to Intelligent Design

Carrier-Free Nanodrugs Reversing Cancer Multidrug Resistance: From Mechanisms to Intelligent Design

Best for: Cancer pharmacology and nanomedicine researchers, medical oncologists and clinical pharmacists, drug design and translational medicine teams, and evidence-based medicine and medical intelligence researchers. Primary keywords: carrier-free nanodrugs; multidrug resistance; P-glycoprotein; self-assembly; synergistic delivery; clinical translation

Abstract / Short Answer

Multidrug resistance (MDR) means tumor cells become cross-resistant to structurally and mechanistically diverse anticancer drugs, driven by P-glycoprotein (P-gp) efflux, enhanced DNA repair, and suppressed apoptosis. Conventional reversal faces a dilemma. Small-molecule P-gp inhibitors such as verapamil, cyclosporine A, and tariquidar cause dose-limiting toxicity because P-gp serves physiological barrier functions at the blood-brain barrier, liver, and kidney. Conventional nanocarriers such as liposomes and polymeric micelles can partly bypass efflux through endocytosis, but drug loading is usually below 10%, so inert carrier material dilutes the active drug and adds immunogenicity and metabolic burden. Carrier-free nanodrugs are formed by self-assembly of pure drug molecules through pi-pi stacking, hydrogen bonding, and electrostatic interaction, with no inert carrier, giving three advantages: near-100% drug loading that produces intracellular concentrations able to overwhelm P-gp efflux; endocytic entry that keeps drug inside vesicles before it reaches the cytosol, avoiding P-gp recognition; and co-assembly of chemotherapeutics with MDR inhibitors for synergistic reversal. This review organizes four mechanism classes (stealth endocytosis, drug overload, mitochondrial targeting, and pathway inhibition), four design dimensions (drug co-assembly, surface functionalization, pH or enzyme-responsive release, and physicochemical tuning), and three representative cases: doxorubicin-curcumin nanoparticles (reversal index about 6.0), paclitaxel-verapamil nanoparticles (82.3% tumor inhibition, 96.8% reduction in lung metastases), and platinum-based carrier-free nanoparticles (reversal index 3.8). Carrier-free nanodrugs are a powerful tool against MDR, but translation is limited by difficulties in tracking in vivo fate, poor batch reproducibility, and scarce long-term safety data, pointing toward CRISPR-based gene-drug co-delivery and single-cell sequencing-guided personalized design. Tools such as QSevidence can support structured evidence checking and synthesis for such reviews.

1. Background: The Clinical Dilemma of MDR and the Case for Carrier-Free Nanodrugs

MDR is the leading cause of chemotherapy failure. Its essence is that tumor cells become cross-resistant to multiple anticancer drugs with different structures and mechanisms, and the resistance mechanisms interact closely. The most studied and clinically significant mechanism is efflux mediated by P-glycoprotein (P-gp), encoded by MDR1. As an ATP-dependent transmembrane transporter, P-gp recognizes and actively pumps anthracyclines, taxanes, and vinca alkaloids out of the cell, lowering intracellular concentration below the therapeutic threshold and making resistant lines persistently refractory. Early reversal strategies focused on small-molecule inhibitors, but verapamil, cyclosporine A, and even the third-generation inhibitor tariquidar showed dose-limiting toxicity in trials, because P-gp performs essential physiological barrier functions at the blood-brain barrier, liver, and kidney, so systemic inhibition disrupts normal drug metabolism and causes neurotoxicity. Conventional carrier-based nanomedicines can partly bypass efflux via endocytosis, but drug loading is usually below 10% by mass, so abundant inert carrier dilutes the active drug and adds immunogenicity and metabolic burden. More critically, the heterogeneity of MDR means blocking a single mechanism is often insufficient, as tumor cells can upregulate other efflux pumps such as ABCG2 and MRP1 or activate alternative resistance pathways.

Carrier-free nanodrugs are a design paradigm aimed at this dilemma: nanoscale delivery systems formed by self-assembly of pure drug molecules through intermolecular interactions such as pi-pi stacking, hydrogen bonding, and electrostatic forces, containing no inert carrier material. They offer three core advantages against MDR. First, ultra-high drug loading, usually near 100%, lets a single nanoparticle carry thousands of drug molecules; after endocytic uptake into resistant cells it releases very high local concentrations of free drug that overwhelm P-gp efflux, achieving concentration-dependent killing. Second, the endocytic route evades efflux: nanoparticles enter through clathrin- or caveolin-mediated endocytosis, and drug molecules remain enclosed in the nanostructure before reaching the cytosol, avoiding recognition and pumping by P-gp at the plasma membrane, the so-called stealth endocytosis mechanism. Third, co-loaded synergistic drugs: two or more agents with synergistic anti-MDR action, such as a chemotherapeutic plus a P-gp inhibitor or apoptosis inducer, can be assembled into a single nanoparticle for spatiotemporally synchronized delivery that inhibits several resistance pathways at once. During evidence preparation for a review, teams can use the QSevidence medical AI tool for dual mechanistic and literature retrieval, using AI guideline retrieval to confirm clinical MDR treatment and medication consensus, and literature evidence appraisal to locate primary studies on P-gp structural biology, endocytic pathways, and mitochondrial apoptosis, building a traceable evidence chain between mechanism and design rather than a simple literature list.

2. Core Mechanisms: Bypassing P-gp Efflux and Intracellular Drug Overload

Carrier-free nanodrugs overcome MDR mainly through a distinct intracellular entry route and drug release kinetics that together circumvent the classical P-gp efflux defense. Free drug enters cells by passive diffusion, and molecules are readily recognized and pumped out by P-gp as they cross the membrane lipid bilayer, leaving intracellular concentrations far below the killing threshold. Carrier-free nanodrugs reshape the physical form and delivery route of the drug, effectively changing the terms of engagement with MDR. Researchers can use QSevidence to archive primary studies by evidence level, separating cell experiments, animal models, and preclinical data, so that in vitro concentration findings are not extrapolated directly into clinical efficacy claims.

Step 1: Bypass P-gp recognition through stealth endocytosis

Carrier-free nanodrugs such as drug nanocrystals and self-assembled nanoparticles are typically 50-200 nm and cannot enter cells by passive diffusion, so they are taken up by endocytosis. This shift is critical: endocytosis, especially when clathrin- or caveolin-mediated, encloses the nanoparticle in a vesicle, so drug molecules remain separated by the lipid bilayer before entering the cytosol and never contact P-gp on the plasma membrane. More importantly, the subcellular distribution of P-gp is highly selective. In several MDR cell lines, P-gp localizes mainly to the plasma membrane and Golgi apparatus, and is not found on endosomal or lysosomal membranes. Once a nanoparticle is endocytosed into the endosomal and lysosomal system, the drug inside sits in a P-gp blind zone. Subsequent lysosomal escape or perinuclear release then creates high drug accumulation in the perinuclear region or cytosol, far from the main site of P-gp action on the inner face of the plasma membrane. By altering subcellular localization, carrier-free nanodrugs cut the interception route of P-gp at its root.

Step 2: Saturate the efflux pump through concentration-driven drug overload

Bypassing efflux is only the first step. True MDR reversal depends on providing intracellular drug concentrations far above those of conventional formulations, thereby overwhelming P-gp kinetically. As an ATP-dependent transporter, P-gp has a maximum transport rate. When intracellular free drug rises sharply above the saturation concentration, the pump cannot export all the molecules in time, net influx increases, and effective killing follows. The ultra-high drug loading of carrier-free nanodrugs is the material basis for this overload effect. Taking doxorubicin-curcumin nanoparticles as an example, a single nanoparticle can carry thousands of drug molecules; after endocytosis it releases rapidly in the acidic lysosomal environment or under cathepsin B action, creating a very high local concentration gradient. Quantitative studies show that compared with free drug solution, carrier-free nanodrugs raise intracellular drug accumulation in resistant cells several-fold to tens of fold, with markedly higher peak concentration and area under the curve. This burst release drives local concentration rapidly to the P-gp saturation threshold, which for some substrates reaches the micromolar range, suppressing efflux and producing concentration-dependent killing. Conventional liposomes or polymeric nanoparticles, by contrast, have low loading, usually below 10%, so the number of drug molecules released from a single particle is limited and a local concentration high enough to saturate P-gp rarely forms, making their MDR reversal weaker than that of carrier-free nanodrugs.

Step 3: Tune delivery efficiency with size, charge, and lysosomal escape

The endocytic efficiency, intracellular trafficking route, and final efficacy of carrier-free nanodrugs are finely controlled by physicochemical properties, with size and surface charge most important. For size, nanoparticles of 50-100 nm enter mainly by caveolin-mediated endocytosis, whereas 100-200 nm particles rely more on clathrin-mediated uptake. The caveolin route is considered more favorable for lysosomal escape, because caveolar vesicles can bypass lysosomal degradation and deliver contents directly to the Golgi or endoplasmic reticulum; designing carrier-free nanodrugs of 60-80 nm may therefore deliver drug to the perinuclear region more effectively and maximize the bypass of P-gp. For surface charge, positively charged nanoparticles bind the negatively charged cell membrane more easily and promote uptake through electrostatic adsorption, but are more likely to remain trapped in lysosomes and face degradation. Negatively charged or zwitterionic surfaces show slower initial uptake but escape lysosomes more readily. For MDR cells, designing an appropriate positive charge, for instance through chitosan modification, to promote uptake while introducing pH-responsive charge reversal or a proton-sponge effect for lysosomal escape, is the key to balancing uptake efficiency and cytosolic release. Lysosomal escape capacity directly determines whether an effective cytosolic drug concentration can form. If nanoparticles remain in lysosomes, drug may be degraded by the acidic environment or recognized and exported by lysosomal efflux transporters such as MRP1. Experimental evidence shows that carrier-free nanodrugs achieving successful lysosomal escape can be several-fold more cytotoxic than counterparts trapped in lysosomes.

3. Design Strategies: Co-Assembly, Surface Functionalization, and Smart Release

Given the complexity of MDR, carrier-free nanodrug design has moved from simple physical mixing to precise, interaction-driven co-assembly with intelligent responsive release. There are three core goals: achieve synergistic anti-MDR effects beyond single drugs through rational pairing and co-assembly; hijack the endocytic route of resistant cells and prolong intracellular retention through surface functionalization; and precisely control release in time and space by exploiting MDR microenvironment features such as acidic pH and overexpressed enzymes, maximizing efflux circumvention.

Drug co-assembly is the starting point. An ideal pair must satisfy two conditions: it can form a stable nanostructure through pi-pi stacking, hydrogen bonding, or electrostatic interaction, and the two agents act synergistically, for example a chemotherapeutic such as doxorubicin or paclitaxel paired with a natural compound such as curcumin or quercetin that inhibits NF-kB signaling or downregulates P-gp. Studies show that co-assembled doxorubicin-curcumin nanoparticles at a specific molar ratio, for example 1:2, raise the reversal index three- to five-fold over the free drug combination in MCF-7/ADR resistant breast cancer cells, attributed to curcumin inhibiting P-gp ATPase activity and the two agents synergistically inducing mitochondrial apoptosis. The critical variables are drug ratio and crystallinity. Co-assembly ratio affects not only particle size and stability but also synergy strength; for example, in a paclitaxel-sorafenib system, when the sorafenib molar ratio exceeds 50%, crystallinity falls and release becomes too fast, reducing penetration depth in resistant tumor spheroids. Design therefore requires phase-diagram analysis to identify the optimal co-assembly window, keeping nanoparticles metastable under physiological conditions but efficiently dissociating inside cells.

Surface functionalization is central to enhancing uptake and retention in resistant cells. One approach is P-gp inhibitor modification: covalently attaching or physically adsorbing verapamil or TPGS on the surface creates a local high-concentration inhibitor microenvironment that transiently suppresses efflux. TPGS-modified doxorubicin nanocrystals showed about 2.5-fold higher uptake than unmodified particles in MCF-7/ADR cells, with intracellular retention extended beyond 6 hours, because TPGS both inhibits P-gp ATPase activity and increases membrane fluidity to promote clathrin-mediated endocytosis. Another approach is tumor-targeting ligand modification: folate or RGD peptide guides nanoparticles into resistant cells through receptor-mediated endocytosis, and because endocytic vesicles bypass membrane efflux pumps to deliver drug near the nucleus, folate-modified paclitaxel-curcumin nanoparticles reduced IC50 roughly four-fold versus non-targeted particles in folate receptor-overexpressing A2780/ADR resistant ovarian cancer cells and achieved deeper spheroid penetration above 100 micrometers via caveolin-mediated endocytosis. Smart responsive release exploits the distinctive biochemistry of the MDR tumor microenvironment, including extracellular pH 6.5-6.8, lysosomal pH 4.5-5.5, and high cathepsin B expression. pH-responsive design using hydrazone or acetal linkages, or exploiting differences in drug pKa, keeps nanoparticles stable at pH 7.4 but releases more than 80% of drug within 30 minutes at lysosomal pH 5.0. Enzyme-responsive design links drug through a peptide specifically cleaved by cathepsin B, such as Gly-Phe-Leu-Gly, to form prodrug-type carrier-free nanoparticles; in MCF-7/ADR cells cumulative release at 24 hours was about 60% higher than a non-responsive control and killing efficiency rose about three-fold, with release occurring only inside lysosomes, fully bypassing extracellular P-gp recognition.

Physicochemical tuning is the fourth design dimension. For size, spherical particles of 50-100 nm generally combine the best enhanced permeability and retention effect with uptake efficiency, but in MDR spheroid models particles below 50 nm penetrate quickly yet are easily pumped out, while 100-200 nm particles penetrate slowly but form a perinuclear drug reservoir through caveolin-mediated endocytosis for sustained release; designing 80-120 nm balances penetration and retention. For shape, rod-like or sheet-like nanoparticles circulate longer and their non-spherical shape induces stronger membrane deformation to promote uptake; rod-shaped paclitaxel nanocrystals with an aspect ratio of about 3 showed 1.8-fold higher uptake than spherical particles in resistant cells, with intracellular retention extended to 8 hours because shape-related delayed lysosomal escape. For surface charge, a cationic charge of +20 to +30 mV enhances membrane binding, but excessive positive charge causes serum protein adsorption and toxicity, so charge-reversal strategies are common: neutral or weakly negative in circulation to avoid opsonization, then positive in the acidic tumor microenvironment to enhance uptake. Together these can be summarized as a four-dimensional framework of co-assembly, active targeting, smart release, and physicochemical optimization. When consolidating these parameters, teams can use the structured evidence generation capability of QSevidence to organize drug pair molar ratios, size ranges, and response thresholds into a reviewable evidence table annotated with the primary source and experimental system for each parameter, supporting design review and reproducibility.

4. Representative Cases and Efficacy Evaluation

By co-assembling chemotherapeutics with resistance inhibitors, carrier-free nanodrugs have shown synergistic antitumor effects in several resistant tumor models. This section examines three representative cases across reversal index, pharmacokinetics, in vivo distribution, and toxicity, summarized below.

Case 1: Doxorubicin-curcumin nanoparticles reversing breast cancer MDR

Nanoparticles self-assembled from doxorubicin (DOX) and curcumin (Cur) through pi-pi stacking and hydrophobic interaction showed marked reversal in MCF-7/ADR cells: free DOX had an IC50 of about 12.5 micromolar, whereas DOX-Cur nanoparticles reduced it to about 2.1 micromolar, giving a reversal index of about 6.0. The mechanism is dual. Curcumin, a known P-gp inhibitor, downregulates P-gp expression and suppresses efflux function, raising intracellular DOX accumulation 3-4 fold, while the nanoparticles enter through clathrin-mediated endocytosis and bypass P-gp efflux, further increasing retention. In xenograft nude mice, tumor growth inhibition reached 78.5%, significantly higher than 45.2% for the free DOX plus Cur combination (P below 0.01). Pharmacokinetics showed tumor DOX concentration remained at 5.8 micrograms per gram 24 hours after dosing versus 1.2 for free drug, indicating enhanced permeability and retention-driven tumor accumulation. Notably, curcumin itself is cardioprotective and partly offsets DOX cardiotoxicity: at the end of treatment, the myocardial apoptosis index in the nanoparticle group was 8.3% versus 21.7% for free DOX, with no significant rise in serum troponin I, showing that the design reduces systemic toxicity through drug-drug synergy while reversing resistance.

Case 2: Paclitaxel-verapamil nanoparticles reversing lung cancer MDR

Carrier-free nanoparticles co-assembled from paclitaxel (PTX) and verapamil (VER) showed distinctive pharmacokinetic advantages in the A549/Taxol model. Compared with free combined dosing, tumor drug accumulation in the nanoparticle group peaked at 12.3 micrograms per gram 4 hours after dosing versus 3.1 for free drug, and half-life extended to 8.5 hours versus 2.1. This difference stems from the size effect, about 120 nm, and surface properties: caveolin-mediated endocytosis avoids P-gp recognition and efflux to produce drug overload. In xenografts, tumor inhibition reached 82.3% versus 51.6% for the free combination (P below 0.001); more notably, lung metastases fell 96.8%, indicating suppression of metastasis as well as primary growth. Mechanistically, verapamil inhibits P-gp efflux to keep intracellular PTX above the effective threshold, while the lysosomal escape capacity of the nanoparticles through pH-responsive release further increases cytosolic bioavailability. For safety, liver toxicity markers ALT and AST were about 40% lower than free drug and no obvious neurotoxicity was observed, consistent with reduced normal-tissue exposure from targeted delivery.

Case 3: Platinum-based nanoparticles overcoming DNA repair-related MDR

Aiming at the enhanced DNA repair, such as the nucleotide excision repair pathway, common in platinum resistance, carrier-free nanoparticles based on cisplatin (CDDP) and oxaliplatin showed distinctive advantages. In A2780/CDDP cells, CDDP nanoparticles had an IC50 of 4.8 micromolar versus 18.2 for free CDDP, a reversal index of 3.8. Mechanistic analysis showed that high-concentration delivery formed abundant platinum-DNA adducts, 12.5 versus 4.2 per million base pairs for free drug, directly saturating the repair capacity of nucleotide excision repair enzymes to overcome DNA repair-mediated resistance. In long-term in vivo toxicity assessment, CDDP nanoparticles markedly reduced cardiotoxicity in 4T1 tumor-bearing mice: serum creatine kinase MB was 55% of free CDDP and the myocardial histopathology score was 1.2 on a 0-4 scale versus 2.8 for free drug (P below 0.05); liver toxicity showed ALT at 62% of free drug with no obvious hepatocellular necrosis or steatosis. These data show that by altering platinum biodistribution, raising the tumor to liver concentration ratio to 3.5 to 1 versus 1.2 to 1 for free drug, the nanoparticles maintain antitumor efficacy while markedly reducing systemic toxicity. The nanoparticles were also active in patient-derived xenograft models of resistant tumors carrying BRCA1 or BRCA2 mutations, suggesting a role after PARP inhibitor resistance in second-line treatment.

Evaluation methodology: from reversal index to spheroid models

The evaluation framework used across these cases provides a standardized basis for preclinical work on carrier-free nanodrugs. In vitro, the reversal index, defined as IC50 in the resistant line divided by IC50 in the sensitive line, is the core measure of reversal, with a value above 2 usually considered clinically meaningful. In vivo, tumor growth inhibition and survival extension, expressed as the median survival time ratio, are the primary endpoints, while drug accumulation such as the tumor to plasma concentration ratio and toxicity markers including troponin I, ALT, and CK-MB are key secondary endpoints. In addition, three-dimensional spheroid models better mimic the resistant tumor microenvironment, with IC50 values typically 2-5 fold higher than in two-dimensional models, so they are recommended as a complement to in vitro validation. When comparing reversal indices across studies, researchers can use QSevidence to check cell line provenance, resistance induction method, and IC50 assay format, since these factors strongly affect comparability and ignoring methodological differences can lead to wrong conclusions.

5. Discussion: Mitochondrial Targeting, Pathway Inhibition, and Limits of In Vivo Fate Tracking

The potential of carrier-free nanodrugs against MDR stems largely from their ability to bypass the upstream efflux barrier represented by P-gp and act directly on the downstream executioner of cell death, the mitochondrion. Conventional chemotherapeutics entering MDR cells are rapidly recognized and pumped out by P-gp, leaving intracellular levels far below the effective threshold. Carrier-free nanodrugs enter through stealth endocytosis, with the nanoscale aggregate keeping drug enclosed in endocytic vesicles and away from direct P-gp contact, and nanoparticles localized in the perinuclear region release drug close to the nucleus, further shortening the distance and time window in which P-gp can capture and export it. Mitochondrial targeting amplifies this advantage. By modifying the surface with lipophilic cations such as triphenylphosphonium or using amphiphilic self-assembly, nanoparticles can selectively interact with anionic phospholipids of the mitochondrial membrane and rupture inside to release drug. The key point is that acting directly on mitochondria bypasses the upstream resistance signaling network built from P-gp overexpression and Bcl-2 upregulation. Mitochondria are the central controller of apoptosis, and loss of membrane potential is an early event that triggers the irreversible cascade of caspase-9 and caspase-3 activation, so even if upstream death receptor pathways are suppressed, mitochondrial-targeted carrier-free nanodrugs can still force apoptosis by directly damaging the mitochondrial membrane and releasing cytochrome c. In MDR breast cancer cells, mitochondrial-targeted nanoparticles reduced spheroid size about 84% more than non-targeted particles, directly demonstrating the value of bypassing upstream signaling to strike the downstream effector.

Co-loading MDR inhibitors is another route. Verapamil, a first-generation P-gp inhibitor, acts as a decoy by competitively binding the substrate site to increase intracellular chemotherapeutic accumulation, but free verapamil reaches effective reversal concentrations in vivo only with cardiotoxicity. Carrier-free nanodrugs co-assemble chemotherapeutic and verapamil at a precise molar ratio for synchronized delivery and synergistic release, producing significant synergistic killing at very low, nanomolar doses far below the micromolar concentrations needed for free drug alone. This nano-synergy lowers the dose and toxicity of a single reversal agent and, through spatial co-localization, ensures both drugs act on the same cell. Multiple reversal agents can also be integrated: for example, a non-immunosuppressive cyclosporine A analog co-loaded with verapamil in one nanoparticle can act on different P-gp binding sites or downstream pathways; in 4T1 tumor-bearing mice, nanoparticles co-delivering paclitaxel and an Akt inhibitor achieved 94.1% tumor inhibition and suppressed 96.8% of lung metastases, demonstrating the potential of multi-target pathway inhibition in vivo.

Moving from laboratory validation toward clinical translation, however, the most critical obstacle is the lack of real-time, high-resolution tracking of in vivo fate, and the limitations appear in three areas. First, imaging modalities are single and sensitivity is insufficient: preclinical work relies mainly on fluorescence imaging, for example exploiting the intrinsic fluorescence of doxorubicin, or radiolabeling to track biodistribution, but a single modality often cannot satisfy both high spatial resolution and high sensitivity. Fluorescence imaging has limited depth penetration and cannot accurately assess mitochondrial accumulation in the core of a solid tumor, while PET and SPECT are sensitive but lack the spatial resolution to distinguish whether nanoparticles sit in the cytosol, in mitochondria, or sequestered in lysosomes. Second, real-time, subcellular dynamic tracking is not achievable: the key to MDR reversal lies in intracellular trafficking and mitochondrial retention kinetics, yet existing techniques such as confocal microscopy usually require fixed and sectioned tissue and cannot follow a single nanoparticle in vivo through endocytosis, escape, and mitochondrial targeting. The high interstitial pressure and dense extracellular matrix of the tumor microenvironment severely limit diffusion, and without real-time tracking the impact of this physical barrier on targeting efficiency cannot be quantified. Third, there are blind spots in long-term safety and biodistribution assessment: because carrier-free nanodrugs are made of pure drug molecules, degradation products are the drug itself, which simplifies metabolism but means mitochondrial-targeting modifications such as triphenylphosphonium can alter intrinsic pharmacokinetics, causing abnormal accumulation in mitochondria of non-target organs such as heart and liver with potential mitochondrial toxicity, yet no technology allows long-term, non-invasive monitoring of nanoparticle retention and clearance in organ mitochondria. In addition, batch-to-batch reproducibility is especially problematic in production because self-assembly is highly sensitive to drug ratio, solvent, and temperature, further increasing variability and interpretive difficulty in tracking results. To overcome these limits, future work should develop multimodal imaging probes integrating fluorescence, photoacoustic, and PET signals in one nanoparticle for multiscale tracking from whole-body distribution to subcellular localization; build in vitro models closer to human physiology, such as microfluidic chips and patient-derived tumor organoids, to provide a benchmark for interpreting in vivo data; and establish standardized GMP-compliant manufacturing to ensure batch consistency. In this process, teams can use the literature appraisal and structured evidence capabilities of QSevidence to organize the sensitivity and resolution parameters of different imaging modalities and the applicable boundaries of various in vitro models into comparative evidence tables, so that methodological choices are grounded in evidence.

6. Conclusion and Outlook: Clinical Translation and Personalized Design

Through their carrier-free nature, carrier-free nanodrugs show potential against cancer multidrug resistance that conventional nanocarrier systems struggle to match. Their core design principles can be summarized in three points: self-assembly between drug molecules achieves ultra-high drug loading so that intracellular concentration transiently exceeds P-gp efflux capacity for concentration-dependent killing; the size effect, typically 50-200 nm, and surface properties bypass P-gp efflux through clathrin- or caveolin-mediated endocytosis so drug molecules avoid recognition; and co-assembly of drugs with different mechanisms achieves synergistic reversal while inhibiting resistance pathways and inducing apoptosis. These principles have been validated in multiple MDR cell lines and animal models.

From proof of concept to clinical application, however, several bottlenecks remain. For scalable manufacturing, carrier-free nanodrugs, especially drug nanocrystals, offer the theoretical advantage of being solvent-free and easy to scale, but poor batch reproducibility is the main barrier: because no carrier material buffers the system, drug crystallinity, polymorph, size distribution, and surface charge are highly sensitive to process parameters, so physicochemical properties and in vivo behavior vary markedly between batches. Converting nanocrystals or self-assembled nanoparticles into stable solid dosage forms such as lyophilized powders also faces challenges of aggregation or polymorphic transition. For in vivo fate tracking and long-term safety, the dissociation, metabolism, and clearance pathways of carrier-free nanodrugs after administration remain unclear; without carrier material, in vivo behavior is governed entirely by the physicochemical properties of the drug molecules and the nanostructure, complicating pharmacokinetic prediction. More importantly, long-term safety data are scarce, as most studies stay at the cell and animal level without systematic assessment of chronic toxicity, immunogenicity, and organ accumulation. In addition, the Cas9 protein itself may trigger humoral and cell-mediated adaptive immune responses, adding an immune safety consideration to CRISPR-based gene-drug co-delivery strategies.

Looking ahead, carrier-free nanodrugs will evolve toward precision, intelligence, and personalization. CRISPR-Cas9-based gene-drug co-delivery is a highly promising direction: co-assembling a chemotherapeutic with an sgRNA and Cas9 ribonucleoprotein complex targeting MDR1 could knock down P-gp expression while delivering chemotherapy, fundamentally reversing resistance, though delivery efficiency, off-target effects, and in vivo editing efficiency remain key challenges. The high loading capacity of carrier-free nanoparticles can accommodate Cas9 protein, sgRNA, and chemotherapeutic simultaneously, but ensuring synchronized release and retained activity requires precise assembly and responsive design. Single-cell sequencing will provide molecular guidance for personalized design: by analyzing transcriptomic features of different resistant subpopulations in a patient's tumor, the dominant resistance mechanism, whether P-gp overexpression, suppressed apoptosis, or enhanced DNA repair, can be identified to guide drug combination and surface ligand design, so that a P-gp-high subpopulation receives nanoparticles co-loading a P-gp inhibitor and chemotherapeutic while a subpopulation with defective mitochondrial apoptosis receives mitochondrial-targeted carrier-free nanodrugs that directly induce apoptosis. Smart responsive release is likewise key: designing nanoparticles that dissociate rapidly in lysosomes or endosomes in response to the MDR microenvironment, low pH and high cathepsin B, can further evade P-gp efflux and enrich drug near the nucleus. Finally, clinical translation must overcome regulatory and intellectual property barriers: because carrier-free nanodrugs often involve co-assembly of two or more drugs, their regulatory path as a fixed-dose combination is unclear and must be clarified as either a new chemical entity or a fixed combination product, while differing patent ownership and licensing agreements may create commercialization barriers. Establishing a standardized quality evaluation system with acceptance criteria for size, drug loading, and in vitro release profiles, and conducting rigorously designed preclinical safety evaluation covering long-term, reproductive, and immune toxicity, will be essential to move carrier-free nanodrugs from bench to bedside. For research teams handling such reviews and translational assessments, QSevidence can play three roles, retrieving mechanistic evidence, annotating evidence levels, and generating structured reviews, consolidating conclusions scattered across nanomaterial, pharmacology, and oncology literature into reviewable evidence tables so that every judgement in design strategy and translation pathway has traceable literature support.

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Medical Disclaimer

This article is based on review literature and preclinical studies. It is provided for medical education, research methodology, and drug development reference only and does not constitute clinical medication advice. The cell and animal findings discussed have not been translated into clinical evidence, and the safety, efficacy, and indications of the nanomedicines involved have not received regulatory approval. They must not be used to guide patient treatment. Clinical decisions must be made by qualified oncologists according to current guidelines and individual patient circumstances.