Overcoming chemotherapy resistance in diffuse large B-cell lymphoma using multifunctional nanocarriers: current advances and clinical challenges

Abstract

Chemoresistance is a primary challenge in diffuse large B-cell lymphoma (DLBCL) treatment. The review is developed to explore the potential of the nanocarrier strategy to minimize resistance in DLBCL treatment. The application of nanocarriers offers promising solutions to increase the drug delivery, avoid resistance, and minimize off-target delivery. Therapeutic strategies of nanocarriers with targeted delivery, gene-based therapies, and combination therapies are explained in the review. This review further evaluates the CXCR4 and Transferrin Receptor 1 (TFR1) efficacy through highly functional nanocarriers that control resistance in the drug pathways. Moreover, it is found that microRNA (miRNA) and small interfering RNA (siRNA) delivery through nanocarriers is beneficial to reduce the chemoresistance and resensitise the DLBCL cells. Furthermore, the review included the significance of multifunctional nanocarriers that combine drug delivery with a stimuli-responsive system and gene regulation that further releases therapeutics in a selective tumour environment. The treatment option is also associated with various challenges, like biological barriers, scalability, safety issues, and the specificity of targeting impacts on the clinical adoption of these technologies. The future research is recommended to focus on the optimization of the nanocarrier design, integration of nanocarriers, and conducting clinical and pre-clinical studies using other therapeutic modalities, that can improve the treatment outcomes for DLBCL patients.

Introduction

Diffuse Large B-Cell Lymphoma (DLBCL) is the most common and aggressive subtype of non-Hodgkin lymphoma (NHL), accounting for approximately 30–40% of all newly diagnosed cases globally [1]. The current standard of care, the R-CHOP regimen (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), has significantly improved survival rates. However, clinical outcomes vary widely. While many patients achieve remission, about 30–40% eventually face relapsed or refractory (R/R) disease, leading to poor prognosis and limited therapeutic options [2].

This therapeutic variability is fundamentally underpinned by the profound molecular heterogeneity of DLBCL, specifically the distinct gene expression profiles of Germinal Center B-cell (GCB) and Activated B-cell (ABC) subtypes, and the presence of high-risk genetic aberrations such as MYC and BCL2 rearrangements. Beyond intrinsic cellular factors, the tumor microenvironment (TME)—characterized by dense extracellular matrix, hypoxia, and immunosuppressive cell infiltration—acts as a critical extrinsic driver of resistance by creating physical barriers to drug penetration and providing survival niches for malignant cells [3]. These diverse resistance mechanisms imply that a uniform systemic chemotherapy approach often fails to achieve deep molecular remission in high-risk subsets. Consequently, there is an urgent need for mechanistically-targeted therapeutic strategies. Multifunctional nanocarriers offer a compelling solution to this gap, as they can be engineered to circumvent physical TME barriers and deliver precise combinations of payloads tailored to specific molecular resistance nodes [4].

Nanocarrier-based solutions offer a compelling rationale to circumvent these biological hurdles. Unlike free drugs, nanocarriers can leverage the enhanced permeability and retention (EPR) effect and active targeting ligands (e.g., anti-CD20) to maximize drug accumulation specifically within tumor cells while sparing healthy tissues [5]. More importantly, recent innovations in stimuli-responsive systems allow specifically for payload release triggered by the acidic or hypoxic conditions of the lymphoma microenvironment, adding a layer of precision to the treatment [6]. By integrating targeted delivery with controlled release, these multifunctional platforms aim to “outsmart” resistance mechanisms at the cellular level.

This mini-review critically synthesizes recent advancements in multifunctional nanocarrier strategies designed to overcome chemotherapy resistance in DLBCL. We explore the intersection of DLBCL biology and nanomedicine, discussing how targeted delivery, gene regulation, and combination therapies can circumvent established resistance pathways. Finally, we address the key translational challenges that must be solved to bring these promising innovations from the bench to the bedside.

Mechanisms of chemotherapy resistance in DLBCL

Chemotherapy resistance in DLBCL is a multifactorial phenomenon. It is not merely a consequence of cellular defense mechanisms but deeply rooted in the molecular heterogeneity of the disease—specifically the distinction between Germinal Center B-cell (GCB) and Activated B-cell (ABC) subtypes—and the complex interplay with the TME. Understanding these biological barriers is a prerequisite for designing effective nanocarrier systems.

The pathophysiology of DLBCL is characterized by profound genetic and epigenetic dysregulation, primarily involving the constitutive activation of B-cell receptor (BCR) signaling and the NF-κB pathway [7]. Genetic hallmarks, such as the translocation or co-expression of MYC and BCL2, create a pro-survival environment that renders cells inherently resistant to standard apoptotic triggers [8]. These molecular drivers are further sustained by an altered metabolic state, including the upregulation of nutrient receptors like Transferrin Receptor 1 (TFR1) to meet high iron demands for proliferation. Consequently, modern targeting strategies aim to exploit these vulnerabilities by utilizing ligands directed at lineage-specific surface antigens (e.g., CD19, CD20, CD79b) or overexpressed chemotactic receptors like CXCR4 [9]. This pathophysiological framework provides the biological “address” for the multifunctional nanocarriers discussed herein, enabling precise delivery that circumvents systemic resistance mechanisms.

Biological drivers of resistance: beyond molecular subtyping

Building upon the clinical variability, the chemoresistance of DLBCL is orchestrated by intricate biological shielding. While the GCB and ABC subtyping provides a prognostic framework, the specific resistance within the ABC subtype is primarily driven by the constitutive activation of the NF-κB pathway, which upregulates anti-apoptotic proteins such as BCL-2 and MCL-1 [10]. Furthermore, the TME interplay mentioned previously is not merely a physical barrier; it functions as a dynamic “pro-survival niche.” Specifically, the secretion of CXCL12 by stromal cells interacts with CXCR4 on lymphoma cells, activating the PI3K/AKT/mTOR axis to circumvent the cytotoxic effects of R-CHOP [11]. By shifting the focus from descriptive subtyping to these specific signaling nodes, we can better define the targets for the multifunctional nanocarriers discussed in the subsequent sections.

Dysregulation of apoptotic pathways and genetic heterogeneity

Even when drugs successfully enter the cell, resistance often stems from the cancer cell’s inability to initiate apoptosis. This is particularly evident in “Double-Hit” or “Triple-Hit” lymphomas, which harbor concurrent rearrangements of MYC and BCL2 and/or BCL6 [12–14].

The anti-apoptotic protein Bcl-2 is highly expressed in approximately 50% of DLBCL cases [15]. It sequesters pro-apoptotic signals, rendering cells insensitive to chemotherapy-induced DNA damage. In the ABC-DLBCL subtype, constitutive activation of the NF-κB pathway drives cell survival and proliferation. This pathway is a key driver of resistance to standard chemotherapeutic agents, as it upregulates downstream survival genes that counteract drug efficacy [16]. Targeting these pathways (e.g., silencing Bcl-2 or blocking NF-κB) represents a critical therapeutic opportunity that nanocarriers can exploit through gene delivery.

The tumor microenvironment (TME) and hypoxia

The physical and chemical landscape of the DLBCL niche contributes significantly to “environment-mediated drug resistance” (EMDR). The rapid proliferation of lymphoma cells often outpaces vascular supply, creating hypoxic regions characterized by low pH and high interstitial fluid pressure [17]. Hypoxic conditions stabilize Hypoxia-Inducible Factor-1α (HIF-1α), which in turn upregulates CXCR4. The CXCL12/CXCR4 axis promotes tumor cell homing to protective niches in the bone marrow or lymph nodes, shielding them from systemic therapy [18]. The dense extracellular matrix (ECM) limits the penetration of therapeutic agents. Nanocarriers must effectively navigate this stiff matrix to reach the deep-seated tumor core [19].

Loss of target antigens (CD20 downregulation)

Since rituximab is the backbone of the R-CHOP regimen, resistance to immunotherapy is equally critical. Long-term exposure to rituximab can lead to CD20 antigen loss, internalization, or polymorphism (e.g., mutations in the MS4A1 gene) [20]. This “antigen escape” renders CD20-targeted therapies ineffective, highlighting the need for nanocarriers that can utilize alternative receptors (such as TFR1 or CXCR4) or deliver payloads without relying solely on CD20 binding [21].

Integrated molecular target landscape: implications for nanocarrier design

The resistance mechanisms described in Sects. 2.1–2.4 form an interconnected signaling network rather than independent barriers. Failure to address one node can activate compensatory pathways and render otherwise effective nanocarrier strategies futile. The key targetable nodes can be organized into four interdependent axes (Fig. 1).

Fig. 1.

Integrated molecular target landscape for nanocarrier-based intervention in DLBCL chemoresistance. Four mechanistically distinct resistance axes are depicted: the MDR efflux axis (P-gp/ABCB1, TFR1, CXCR4), the apoptotic suppression axis (BCL-2, MYC/BRD4, BTK/PI3K/NF-κB), the TME-mediated resistance axis (HIF-1α, ECM barrier, low pH/high GSH), and the metabolic reprogramming axis (PDK4, CD20 loss, rituximab resistance). Teal boxes indicate the corresponding nanocarrier-based intervention strategy for each axis. Grey dashed boxes highlight cross-axis convergence nodes—molecular intersections where targeting a single node affects multiple axes simultaneously. Subtype-specific axis dominance is annotated at the bottom

The first is the MDR efflux axis, anchored by P-gp/ABCB1. Because P-gp expels hydrophobic drugs via passive membrane diffusion, nanocarriers entering cells through receptor-mediated endocytosis—via TFR1 or CXCR4—circumvent this mechanism entirely while simultaneously exploiting receptors overexpressed in resistant DLBCL clones [1].

The second is the apoptotic suppression axis, encompassing BCL-2, MYC/BCL2/BCL6 rearrangements, and constitutive NF-κB activation in ABC-DLBCL [22]. BCL-2 silencing via siRNA-loaded nanocarriers restores the apoptotic threshold in combination with DNA-damaging agents. For MYC—structurally undruggable—indirect targeting via BRD4 inhibition offers a viable alternative: nanocarrier-mediated siBRD4 delivery has demonstrated simultaneous suppression of MYC and downstream survival genes in preclinical models [23]. NF-κB activation in ABC-DLBCL is further amplified by constitutive BCR signaling through BTK and PI3K/AKT [24]. Since acquired BTK inhibitor resistance frequently involves PI3K/AKT/mTOR pathway compensation [18], nanocarriers co-delivering BTK inhibitors alongside BCL-2 silencing agents offer a mechanistic rationale for blocking both the primary pathway and its escape route simultaneously.

The third is the TME-mediated resistance axis, driven by HIF-1α stabilization, CXCR4-mediated tumor sanctuary homing, and ECM-imposed drug penetration barriers [25]. Stimuli-responsive nanocarriers exploiting low pH or elevated glutathione as release triggers directly convert the hostile TME into a precision drug-release mechanism [26].

The fourth is the metabolic reprogramming axis, exemplified by PDK4-driven suppression of CD20 expression and rituximab resistance. Exosome-mediated siPDK4 co-delivery with anti-CD20 therapy directly targets this axis, restoring chemosensitivity in rituximab-refractory cells [27].

Critically, these axes converge at shared molecular nodes: BCR/NF-κB activation simultaneously drives apoptotic suppression and metabolic reprogramming; HIF-1α-mediated CXCR4 upregulation links hypoxic TME to tumor homing; and MYC rearrangement amplifies both BCL-2-dependent survival and metabolic rewiring. This convergence underscores the superior therapeutic rationale of multi-payload nanocarriers capable of targeting two or more axes simultaneously over single-agent approaches.

Nanocarrier strategies to overcome chemoresistance in DLBCL

The application of nanomedicine in DLBCL is not merely about improving drug solubility; it represents a strategic intervention to circumvent the biological barriers identified in the previous section. By engineering carriers with specific physicochemical properties, researchers can exploit distinct mechanisms—ranging from active targeting to gene silencing—to restore chemosensitivity in refractory lymphoma cells.

Active targeting: circumventing efflux pumps via receptor-mediated endocytosis

One of the most effective strategies to circumvent P-gp-mediated drug efflux is to alter the entry route of chemotherapeutics. While free drugs enter via passive diffusion and are easily pumped out, nanocarriers modified with specific ligands can enter cells via receptor-mediated endocytosis, a pathway that often circumvents membrane pumps entirely.

While anti-CD20 (rituximab) is the gold standard targeting ligand, its utility is limited in R/R patients who have experienced CD20 antigen loss. Consequently, recent research has pivoted towards alternative receptors. Transferrin Receptor 1 (TFR1/CD71) has emerged as a particularly compelling alternative target in DLBCL. The receptor mediates clathrin-dependent endocytosis of iron-bound transferrin to meet the elevated iron demands of rapidly proliferating tumor cells, and its surface expression is consequently upregulated severalfold in DLBCL relative to resting lymphocytes—with dataset analyses confirming that high TFR1 expression is specifically associated with high-risk disease [28]. This differential expression creates a favorable therapeutic window: nanocarriers conjugated with transferrin exploit TFR1-mediated endocytosis to achieve intracellular drug delivery that circumvents membrane-bound efflux transporters, effectively circumventing one of the primary mechanisms of chemoresistance in DLBCL. The CND-Dox-TF platform demonstrated 10–100-fold greater potency than free doxorubicin against DLBCL cell lines in a TFR1-dependent manner confirmed by gain- and loss-of-function studies, and improved overall survival in patient-derived xenograft models when substituted for conventional doxorubicin within the R-CHOP backbone [28]. Critically, TFR1 targeting remains functional in CD20-negative relapsed/refractory disease, addressing a major vulnerability of rituximab-based regimens.

The CXCL12/CXCR4 axis represents a mechanistically distinct but equally important target, and CXCR4-targeted nanocarriers offer a dual therapeutic benefit that extends beyond simple receptor-mediated drug delivery. Under hypoxic TME conditions, HIF-1α transcriptionally upregulates CXCR4 on DLBCL cells, which then migrate along CXCL12 gradients secreted by bone marrow mesenchymal stromal cells toward the perivascular niche. Within this sanctuary, stromal contact activates PI3K/MAPK survival signaling, maintains tumor cells in a quiescent, chemotherapy-resistant state, and constitutively suppresses apoptosis—a mechanism that underlies both treatment failure and minimal residual disease persistence in bone marrow-involved DLBCL [29]. CXCR4-targeted nanocarriers disrupt this protection on two simultaneous levels: the targeting ligand occupies and functionally blocks CXCR4, preventing further CXCL12-driven homing and stripping tumor cells of niche-derived survival signals, while the encapsulated cytotoxic payload is delivered intracellularly to cells that are now exposed and chemosensitized [30]. In a DLBCL disseminated mouse model, the T22-peptide-functionalized protein nanocarrier T22-GFP-H6 demonstrated selective accumulation in bone marrow and lymph node compartments harboring CXCR4-overexpressing tumor cells, with cytotoxic activity confined to malignant cells and no histological toxicity to normal marrow—a selectivity attributable to the higher CXCR4 expression on DLBCL cells compared to resident hematopoietic progenitors [31]. This dual functionality—simultaneous niche disruption and targeted payload delivery—distinguishes CXCR4-targeted nanocarriers from conventional chemotherapy and positions them as particularly suited to the treatment of bone marrow-involved or relapsed DLBCL.

Strategies for CD20-negative disease

Engineering nanocarriers to function independently of CD20 expression represents an active area of nanocarrier design, and several complementary strategies have been developed to address this vulnerability.

The most direct approach is alternative receptor targeting—substituting CD20 as the targeting ligand with receptors whose expression is maintained or upregulated regardless of prior rituximab exposure. TFR1 and CXCR4, discussed in detail in Sect. 3.1, exemplify this strategy: both are overexpressed in DLBCL through mechanisms intrinsic to tumor cell metabolism and microenvironmental signaling rather than through immunotherapy selection pressure, making antigen loss under rituximab treatment functionally irrelevant to their targeting utility [28].

A second strategy exploits the physicochemical properties of the DLBCL TME rather than any specific tumor-surface antigen. The acidic interstitial pH (6.5–6.8) generated by Warburg-effect metabolism and the hypoxic conditions characteristic of rapidly proliferating lymphoma masses can be harnessed to trigger drug release selectively at the tumor site. pH-responsive nanocarriers incorporating acid-labile linkers—such as hydrazone bonds or imino groups—are stable at physiological pH during circulation but undergo rapid hydrolysis and payload release upon encountering the acidic TME, achieving tumor selectivity without requiring any surface receptor interaction [32]. Hypoxia-responsive carriers exploiting azoreductase-cleavable nitroimidazole groups provide an analogous antigen-independent release mechanism operative specifically within hypoxic tumor regions.

A third approach employs biomimetic cell membrane coating, in which nanocarrier surfaces are functionalized with membranes derived from autologous lymphoma cells or immune cells. Cancer cell membrane-coated nanoparticles inherit the homotypic adhesion molecules of their source cells, enabling selective binding to tumor cells of the same origin through surface adhesion mechanisms that are entirely CD20-independent [33]. Macrophage membrane-coated variants exploit the inherent inflammatory tumor-homing capacity of macrophage surface proteins to achieve TME-directed accumulation without relying on any specific tumor antigen [34]. Collectively, these strategies decouple nanocarrier tumor selectivity from antigen expression status, providing a rational design framework for next-generation platforms capable of maintaining efficacy in CD20-negative relapsed DLBCL.

Gene-based resensitization: silencing the “undruggable”

For DLBCL cases driven by “undruggable” anti-apoptotic proteins (e.g., Bcl-2, Myc), gene-based nanotherapies offer a precision tool to restore apoptotic signaling. Small interfering RNA (siRNA) and microRNA (miRNA) are potent modulators but are rapidly degraded in the bloodstream. Cationic liposomes and polymer-based nanoparticles (e.g., PEI, chitosan) have been extensively employed to protect these nucleic acids and facilitate endosomal escape. These therapies assist in the targeted delivery of RNA-based therapies like miRNA and siRNA into the tumor cells (Fig. 2). A notable approach involves the delivery of siPDK4 (targeting Pyruvate Dehydrogenase Kinase 4) alongside anti-CD20 therapy [35]. This strategy reverses the metabolic reprogramming associated with rituximab resistance, effectively resensitizing the tumor cells [27]. Emerging platforms are now exploring the delivery of CRISPR/Cas9 systems to permanently disrupt resistance genes. Although still in nascent stages for DLBCL, this approach holds the potential for a “one-and-done” reversal of chemoresistance [36, 37]. The major bottleneck for gene-based nanocarrier therapy remains endosomal escape: following receptor-mediated endocytosis, the majority of internalized carriers are trafficked to acidifying endolysosomes where nucleic acid payloads are enzymatically degraded before cytosolic release can occur. Several chemical strategies have been developed to overcome this barrier, each exploiting distinct mechanisms [38].

Fig. 2.

Schematic representation of the four key axes for overcoming chemoresistance in DLBCL using multifunctional nanocarriers. DLBCL, Diffuse large B-cell lymphoma; MDR, multidrug resistance; TME, tumor microenvironment; P-gp, P-glycoprotein; siRNA, small interfering RNA

The most extensively studied approach is the proton sponge effect, exhibited by cationic polymers with high buffering capacity such as polyethylenimine (PEI) and PAMAM dendrimers. As endosomal pH drops from ~ 6.5 to ~ 5.0 during maturation, these polymers sequester protons, sustaining V-ATPase-driven proton pumping and driving secondary influx of Cl⁻ ions and water—ultimately generating osmotic pressure sufficient to rupture the endosomal membrane and release the genetic payload into the cytosol [39]. While PEI remains the benchmark proton sponge material, its clinical utility is constrained by dose-dependent cytotoxicity arising from membrane disruption in non-target compartments; low-molecular-weight PEI derivatives and imidazole-containing polymers such as polyhistidine have therefore been developed to preserve buffering capacity while reducing toxicity [40]. A mechanistically distinct strategy employs ionizable lipids—the basis of clinically approved LNP formulations—which are neutral at physiological pH but become cationic within acidifying endosomes, promoting electrostatic interaction with anionic endosomal membrane lipids, phase separation, and transient pore formation that enables nucleic acid release. A third approach utilizes fusogenic peptides such as GALA and melittin derivatives, which adopt pH-dependent amphipathic α-helical conformations in the acidic endosomal environment and directly disrupt the lipid bilayer through membrane insertion and pore formation. In the DLBCL context, where siRNA payloads targeting BCL-2, PDK4, or BRD4 must reach the cytosol intact to engage the RISC machinery, rational selection among these strategies—balancing escape efficiency against cytotoxicity and immunogenicity—represents a critical design parameter for next-generation gene-based nanocarriers. Additionally, off-target gene silencing remains a safety concern that requires rigorous validation.

Synergistic co-delivery: the “one-stone, two-birds” approach

Multidrug resistance often requires a multi-pronged attack. Co-delivery systems encapsulate a chemotherapeutic agent (e.g., doxorubicin or vincristine) alongside a chemosensitizer (e.g., Bcl-2 inhibitor or siRNA) within a single polymeric micelle or lipid nanoparticle [41, 42].

This ensures that both agents reach the tumor cell simultaneously and at a precise synergistic ratio, which is difficult to achieve with separate systemic administrations. For example, co-delivering a Bcl-2 inhibitor with a DNA-damaging agent can lower the apoptotic threshold, ensuring that the DNA damage actually leads to cell death rather than repair. The combination therapies are based on the co-delivery of various therapeutic agents through single nanocarriers that can increase the therapeutic effects through synergistic mechanisms (Fig. 3). A key challenge in co-delivery is the differing physicochemical properties of the payloads (e.g., hydrophobic drugs vs. hydrophilic siRNA), which complicates the loading process and stability of the carrier.

Fig. 3.

Rational design of multifunctional nanocarriers to overcome chemoresistance in Diffuse Large B-Cell Lymphoma (DLBCL). The schematic illustrates the paradigm shift from conventional treatment challenges to advanced nanomedicine strategies. These integrated strategies aim to maximize intra-tumoral drug accumulation, minimize off-target effects, and ultimately resensitize DLBCL cells to treatment

Stimuli-responsive systems: exploiting the TME

To minimize systemic toxicity—a major limitation of R-CHOP in elderly patients—“smart” nanocarriers are designed to release their payload only in response to specific TME triggers. The acidic microenvironment (pH ~ 6.5) of DLBCL tissues can trigger the disassembly of pH-sensitive polymers, releasing the drug specifically at the tumor site. Similarly, the high concentration of glutathione (GSH) in the cytoplasm of cancer cells can be exploited by disulfide-linked nanocarriers, which remain stable in the blood but rapidly degrade to release drugs once inside the cancer cell [43, 44]. It ensures the release of the drug preferably in the tumor cells or in the areas where an active resistance mechanism is present. In addition, redox-sensitive nanocarriers also release drugs while responding to higher oxidative stress present in the cancer cells. It also involves the release due to ligand trigger applied in combination with targeted delivery approaches.

Exosome-based biomimetic carriers

Unlike synthetic carriers, exosomes are naturally derived extracellular vesicles that possess innate biocompatibility and low immunogenicity. For example, PDK4 siRNA plus anti-CD20 is delivered through exosomes (Table 1).

Table 1.

Nanocarrier strategies in overcoming chemoresistance in DLBCL

Strategy	Mechanism	Example/approach	References
Targeted delivery	Nanocarriers with ligands targeting specific receptors on tumor cells	Transferrin Receptor 1 (TFR1)–Mediated Delivery using CND-linked Dox + transferrin to target TFR1	[28]
Gene-based therapies	Delivery of siRNA, miRNA, or CRISPR/Cas9 to silence resistance genes	Exosome nanoparticle therapy delivering siPDK4 + anti-CD20 to reverse rituximab resistance	[35]
Combination therapies	Co-delivery of chemotherapeutics and gene regulators to enhance efficacy	Co-encapsulation of Bcl-2 inhibitors (siRNA or drugs) with chemotherapy to restore apoptosis pathways	[49]
Stimuli-responsive systems	Release of drugs in response to environmental changes (pH, redox, ligands)	pH-sensitive or redox-sensitive nanocarriers for targeted drug release in the TME	[50, 51]

The capacity of exosomes to traverse formidable biological barriers, such as the blood-brain barrier (BBB), has been mechanistically elucidated by Zhang et al. [17]. Rather than merely relying on the innate homing abilities of extracellular vesicles, the study demonstrated that functionalizing exosomes with targeting ligands (e.g., c(RGDyK) peptides) allows them to exploit receptor-mediated transcytosis, significantly enhancing the delivery of therapeutic payloads to the brain parenchyma in lymphoma models. However, a critical evaluation of this strategy reveals a complex trade-off: while surface engineering improves specificity, it may also alter the exosomal ‘biomolecular corona’ in systemic circulation, potentially leading to accelerated hepatic clearance. Furthermore, the heterogeneity of exosomal membranes remains a challenge for achieving consistent BBB penetration rates across different patient cohorts. Therefore, the translation of such biomimetic platforms requires not only a focus on homing efficiency but also rigorous characterization of their colloidal stability and long-term immunogenicity within the tumor microenvironment. For example, Bor and Hosta-Rigau explained that siPDK4 is also engineered to deliver therapeutic drugs to reduce resistance and restore sensitivity. Despite their promise, exosome therapy faces significant hurdles in scalability. Producing clinical-grade exosomes with consistent batch-to-batch quality is currently much more difficult and expensive than manufacturing synthetic liposomes.

However, a critical immunological caveat must be acknowledged when considering tumor cell-derived exosomes (TEXs) as therapeutic carriers. Unlike exosomes derived from healthy donor cells, TEXs carry a cargo profile that directly reflects the malignant state of their parent cell—a property that renders the broad characterization of exosomes as “low immunogenicity” vehicles potentially misleading in this context. DLBCL-derived exosomes have been shown to express oncogenic surface proteins including c-Myc, Bcl-2, and CD20, and to act primarily as immunosuppressive mediators in vitro by inducing T cell apoptosis and upregulating PD-1 on effector T cells, effects associated with accelerated tumor growth in vivo. This DLBCL-specific observation is consistent with the broader mechanistic evidence that TEXs carry surface PD-L1 in the same membrane topology as cell-surface PD-L1, enabling systemic CD8+ T cell suppression through direct PD-1 ligation—a mechanism operating not only within the TME but also in draining lymph nodes and peripheral circulation [45]. In DLBCL patients, elevated plasma exosomal PD-L1 has been significantly correlated with higher IPI scores, non-GCB subtype, and advanced Lugano stage, underscoring the clinical relevance of TEX-mediated immunosuppression to disease progression and prognosis [46]. Beyond PD-L1, TEXs carry TGF-β, FasL, and immunosuppressive miRNAs that collectively suppress NK cell cytotoxicity, promote Treg expansion, and impair dendritic cell maturation [47].

For exosome-based therapeutic platforms in DLBCL, these findings raise two practical concerns. First, therapeutic exosomes derived from DLBCL tumor cells—even when engineered to carry siRNA or cytotoxic payloads—may retain residual immunosuppressive surface cargo that inadvertently reinforces immune evasion in the recipient. Second, since exosomal PD-L1 has been shown to confer resistance to anti-PD-1 checkpoint therapy even in tumors incapable of secreting their own exosomes, systemically administered TEX-based carriers could theoretically compromise concurrent immunotherapy. To address these risks, future platforms should prioritize non-tumor cell sources—such as dendritic cells, mesenchymal stem cells, or autologous T cells—or employ surface engineering to remove immunosuppressive ligands prior to therapeutic loading. Immunological profiling of exosome batches, including PD-L1 quantification and immune cell functional assays, should be incorporated as a standard quality attribute in preclinical development [48].

The clinical translation of exosome-based carriers is strictly contingent upon the standardization of isolation and purification processes. The recently released MISEV2023 guidelines [52] emphasize the necessity of rigorous reporting regarding EV separation and characterization to ensure batch-to-batch reproducibility. Currently, ultracentrifugation (UC) remains the predominant research standard due to its capacity for large-volume processing; however, its inherent limitations, including low purity (co-isolation of protein aggregates) and potential structural damage caused by high shear forces, hinder its clinical utility [53]. In contrast, microfluidics offers superior precision and high-purity isolation by leveraging physical or immunoaffinity properties at the microscale, yet it currently lacks the throughput required for industrial-scale manufacturing [54]. For clinical-grade production, tangential flow filtration (TFF) has emerged as a more scalable and gentler alternative, demonstrating a 7-fold higher yield of biologically active exosomes compared to conventional UC-based methods [55].

The paradigm of multifunctionality: integrated design principles

To effectively circumvent the multifaceted resistance of DLBCL, the development of nanocarriers has shifted from single-functional platforms to integrated multifunctional systems. Multifunctionality is defined by the synergistic consolidation of active targeting, co-delivery of heterogeneous payloads, and stimuli-responsive drug release within a single nanostructure [56]. Such “all-in-one” designs are essential for addressing the biological complexity of lymphoma; for instance, a nanocarrier can be engineered to target CXCR4 for TME penetration while simultaneously co-delivering doxorubicin and siRNA to silence BCL2, triggered specifically by the acidic endosomal pH [57]. This integrated approach ensures spatiotemporal control over drug pharmacokinetics, minimizing systemic exposure while maximizing the synergistic suppression of divergent resistance pathways [58].

Challenges and limitations

While the preclinical results of multifunctional nanocarriers in DLBCL are encouraging, the transition from bench to bedside remains fraught with challenges. The discrepancy between robust in vivo efficacy in murine models and the modest success rates in clinical trials highlights several critical hurdles that must be addressed.

Manufacturing and scalability (CMC challenges)

A major bottleneck is the complexity of “multifunctional” designs. While adding targeting ligands, pH-sensitive linkers, and co-loaded drugs enhances efficacy in the lab, it exponentially increases the difficulty of Good Manufacturing Practice (GMP) production. Synthesizing complex nanostructures (e.g., ligand-modified polymeric micelles) with precise reproducibility at a large scale is technically demanding. Slight variations in size or surface charge can drastically alter pharmacokinetics. For emerging exosome-based therapies, the lack of standardized isolation and purification protocols leads to significant heterogeneity. Achieving high-yield production of clinical-grade exosomes without contamination remains an unresolved engineering challenge [59].

These manufacturing complexities carry profound economic implications that represent a formidable barrier to widespread clinical adoption. The cost of producing multifunctional nanocarriers under GMP conditions substantially exceeds that of conventional chemotherapeutic agents. A complete drug development program for a nanomedicine reaching the clinic has been estimated to require a minimum investment of $350 million USD, with scale-up and GMP compliance constituting the most cost-intensive phases [60]. Even for the most clinically established nanocarrier in lymphoma—liposomal doxorubicin (e.g., Doxil^®/Caelyx^®)—manufacturing costs are considerably higher than those of the free parent drug, a disparity that directly translates into higher drug acquisition costs for healthcare systems [61]. For next-generation actively targeted nanocarriers, such as CXCR4-functionalized nanoparticles or siRNA-loaded exosomes, per-cycle costs are expected to be substantially higher still, given the additional manufacturing steps of ligand conjugation, nucleic acid synthesis, and multi-parameter quality control characterization required at each production stage. Notably, the European Medicines Agency (EMA) has reported that approximately 30% of nanomedicine regulatory applications fail due to inadequate manufacturing data, reflecting the disproportionate regulatory burden that complex nanocarrier products face relative to small-molecule drugs [62].

From a health economics perspective, the cost-effectiveness of nanocarrier-based therapies in DLBCL will ultimately hinge on their ability to generate meaningful improvements in patient outcomes that justify the higher upfront investment. In the DLBCL context, cost-effectiveness analyses of novel combination regimens provide a useful benchmark: for instance, a pharmacoeconomic model evaluating R²-CHOP versus R-CHOP in the Chinese healthcare system reported an incremental cost-effectiveness ratio (ICER) of $35,159 per QALY [63], which approached but did not clearly exceed the willingness-to-pay threshold. This suggests that even modest improvements in efficacy can be economically justifiable in this indication—a finding that supports the theoretical value proposition of resistance-targeting nanocarriers in the R/R DLBCL setting, where the costs of subsequent salvage therapies, stem cell transplantation, and CAR-T cell therapy are themselves substantial. However, this remains a theoretical argument until Phase II/III clinical data are available. To bridge this gap, future development programs should prioritize cost-reduction strategies alongside efficacy optimization. Emerging manufacturing technologies—including microfluidics-based continuous production and particle replication in non-wetting templates (PRINT)—have demonstrated the potential to reduce batch-to-batch variability while significantly lowering per-unit production costs, offering a plausible pathway toward economically viable large-scale nanocarrier manufacturing [64].

The “EPR effect” controversy and biological barriers

The EPR effect—the cornerstone of passive nanocarrier tumor targeting—is predicated on leaky tumor vasculature and impaired lymphatic drainage that allow nanoparticles to accumulate preferentially at tumor sites. However, the biological conditions underpinning the EPR effect differ fundamentally between murine models and human patients, and this discrepancy has been a primary driver of clinical translation failures. In subcutaneous mouse xenografts, rapidly induced angiogenesis generates highly permeable, structurally immature vessels with abundant endothelial fenestrations, producing an EPR effect that is artificially amplified relative to the clinical setting. Human tumors, by contrast, display significant inter- and intra-patient variability in vascular architecture, with heterogeneous fenestration density, higher pericyte coverage, elevated interstitial fluid pressure (IFP), and a denser ECM—all of which collectively restrict nanoparticle extravasation and intratumoral penetration [65]. Meta-analyses of preclinical data have demonstrated that a median of only approximately 0.7% of the injected nanoparticle dose actually reaches solid tumors in humans, and clinical imaging studies suggest that detectable EPR-mediated accumulation is observed in only 20–30% of patients [66].

In DLBCL specifically, the EPR landscape is further complicated by the disseminated, non-solid nature of the disease. Unlike solid tumors, DLBCL primarily involves lymph nodes, bone marrow, and extranodal sites—compartments where the vascular architecture consists of sinusoidal vessels with discontinuous endothelia rather than the fenestrated neovasculature that drives conventional EPR [67]. While this sinusoidal structure allows passive nanocarrier access to some extent, it does not generate the sustained retention characteristic of EPR in solid tumor models, and it varies significantly across involved organ sites and disease stage. Consequently, passively targeted nanocarriers that demonstrated robust tumor accumulation in subcutaneous DLBCL xenografts cannot be presumed to achieve equivalent delivery in the disseminated nodal and marrow compartments of clinical patients. This mechanistic gap explains why active receptor-mediated targeting—via TFR1, CXCR4, or anti-CD20 ligands—is not merely an enhancement over passive delivery in DLBCL, but a functional necessity for reliable intracellular drug accumulation in this disease. PEG-mediated stealth circulation, while useful for extending nanocarrier half-life, cannot compensate for fundamentally inadequate EPR-driven tissue extravasation, and repeated dosing carries the additional risk of anti-PEG antibody-mediated accelerated blood clearance.

The immunological basis of this risk warrants explicit discussion in the DLBCL context. PEGylated nanocarriers function as thymus-independent type-2 (TI-2) antigens, cross-linking B-cell receptors on splenic marginal zone B cells and driving T cell-independent anti-PEG IgM production following first-dose administration [68]. This anti-PEG IgM opsonizes subsequent doses, activating complement (C3a/C5a) and triggering rapid mononuclear phagocyte system (MPS) clearance—the ABC phenomenon—which dramatically shortens nanocarrier half-life and redirects drug accumulation from tumor tissue to the liver [69]. The clinical relevance is amplified by the finding that pre-existing anti-PEG antibodies are detectable in over 25% of the general population due to ubiquitous PEG exposure through pharmaceutical excipients, cosmetics, and food additives, meaning a substantial proportion of DLBCL patients may exhibit ABC responses even upon first nanocarrier administration [70].

In the DLBCL treatment setting specifically, this immunological barrier is compounded by standard supportive care. Pegfilgrastim (PEG-G-CSF) is routinely co-administered after each R-CHOP cycle for neutropenia prophylaxis, and clinical data in hematological patients confirm that this agent itself induces and amplifies anti-PEG IgM responses in a dose-dependent manner [71]. Any PEGylated nanocarrier introduced into this treatment context would therefore face a progressively deteriorating pharmacokinetic profile across the six standard R-CHOP cycles, with each successive dose cleared more rapidly than the last. The ABC phenomenon is most pronounced at dosing intervals of 3–7 days and attenuates only when intervals exceed two weeks [72]—a constraint that conflicts directly with mid-cycle nanocarrier dosing strategies. To address this, future DLBCL nanocarrier development should prioritize PEG-free surface coatings—such as polyglycerol, zwitterionic polymers, or biomimetic cell-membrane coatings—or incorporate baseline anti-PEG antibody screening as a patient stratification criterion in clinical trial design.

Safety concerns: immunogenicity and off-target editing

The safety profile of gene-editing nanocarriers requires rigorous scrutiny. While CRISPR holds promise for permanently silencing resistance genes, the risk of off-target editing and chromosomal rearrangements poses a significant safety concern for long-term clinical application [73]. Systemic administration of viral vectors or cationic lipids can trigger cytokine release syndrome (CRS), a particular concern in lymphoma patients whose immune systems are already dysregulated [27, 74].

The integration of nanotechnology with adoptive cell therapy (ACT) represents a frontier strategy to overcome the immunosuppressive microenvironment of DLBCL. Contemporary ACT modalities—including tumor-infiltrating lymphocytes (TILs), chimeric antigen receptor (CAR) T-cells, and natural killer (NK) cells—have demonstrated clinical efficacy in hematological malignancies but remain limited by poor tumor infiltration and ex vivo expansion hurdles [75]. Nanocarriers can be engineered to deliver cytokines or metabolic modulators directly to infused immune cells, thereby enhancing their persistence and cytotoxic activity. For example, nanomedicine-mediated “backpacking” of ACT products allows for the localized release of stimuli, mitigating systemic toxicities while promoting the eradication of chemotherapy-resistant lymphoma cells [75–78].

Precision nanomedicine strategy

To overcome these barriers, the field must pivot from a “one-size-fits-all” approach to precision nanomedicine. Future clinical trials should incorporate biomarkers (e.g., TFR1 expression levels) to screen for patients most likely to benefit from targeted nanocarriers. Shifting focus towards “self” carriers (e.g., patient-derived exosomes or cell-membrane-coated nanoparticles) may offer a solution to the immunogenicity issues associated with synthetic polymers. A comprehensive summary of these translational hurdles and the corresponding research priorities is presented in Table 2.

Table 2.

Challenges and future directions in nanocarrier development for DLBCL

Challenge/direction	Description	References
Biological barriers	Difficulty in crossing biological barriers, such as the blood-brain barrier and TME	[79]
Safety and toxicity concerns	Potential for immune responses or off-target toxicity	[80]
Manufacturing and scalability	Issues in producing nanocarriers at a clinical scale with consistent quality	[81]
Targeting specificity	Precise targeting of nanocarriers to heterogeneous tumor cells	[82]
Clinical translation gaps	Barriers to regulatory approval and patient-specific factors delaying clinical trials	[83]
Advanced design of multifunctional nanocarriers	Focus on integrating multiple functions in a single carrier for enhanced efficacy	[84]
In vivo/pre-clinical studies	Robust pre-clinical validation to assess safety, efficacy, and pharmacokinetics	[85]
Biomarker-guided delivery	Biomarkers guide precise delivery and monitor therapeutic effects	[86]

A foundational principle of personalized nanomedicine in DLBCL is that the molecular subtype of a patient’s disease should directly inform the choice of nanocarrier strategy. The two major cell-of-origin (COO) subtypes—germinal center B-cell (GCB) and activated B-cell (ABC)—differ profoundly not only in prognosis but also in their dominant oncogenic pathways, and these differences have direct implications for nanocarrier target selection [87]. ABC-DLBCL, which accounts for approximately 50% of cases and is characterized by constitutive NF-κB activation and BCR signaling pathway mutations, is associated with a significantly worse 3-year progression-free survival following R-CHOP (~ 40%) compared to GCB-DLBCL (~ 75%) [3]. This disparity underscores the inadequacy of a uniform nanocarrier approach: an NF-κB-silencing siRNA nanocarrier may offer substantial benefit in ABC-DLBCL, where NF-κB is the dominant driver of chemoresistance, yet be largely irrelevant in GCB-DLBCL, where BCL-2 overexpression and MYC rearrangements represent the primary therapeutic vulnerabilities. Similarly, the recently approved polatuzumab vedotin-based regimen (pola-R-CHP) demonstrated a markedly superior hazard ratio for disease progression in the ABC subtype (HR 0.34) compared to the GCB subtype (HR 1.18) in the Phase III POLARIX trial, providing compelling clinical proof that subtype-specific molecular targeting is both feasible and clinically meaningful. For nanocarrier developers, this mandates that COO profiling—using validated platforms such as the NanoString Lymph2Cx assay or digital gene expression tools—be incorporated as a prerequisite in future nanocarrier clinical trial design, rather than treating all DLBCL patients as a homogeneous population. Furthermore, patients harboring MYC/BCL-2 double-hit or triple-hit rearrangements represent a particularly high-risk subgroup in which standard R-CHOP is consistently insufficient [88]; for these patients, nanocarriers co-delivering BCL-2 inhibitors alongside DNA-damaging agents may represent the most rationally designed therapeutic strategy.

Beyond inter-patient molecular subtype differences, intra-tumoral heterogeneity (ITH) poses a specific and underappreciated challenge for actively targeted nanocarriers. Studies in B-cell malignancies have demonstrated that spatially separated tumor deposits within the same patient can harbor divergent mutation profiles, with the majority of single nucleotide variants being site-exclusive rather than shared across lesions [18]. This spatial heterogeneity has a direct implication for receptor-targeted nanocarriers: if a targeting ligand (e.g., anti-TFR1 or CXCR4 peptide) binds to a receptor expressed on the dominant tumor clone but not on minority subclones, those resistant subclones will be spared and subsequently expand under selective pressure—a phenomenon analogous to the “antigen escape” observed with CD20-targeted rituximab. As single-cell sequencing technologies increasingly reveal the clonal architecture of individual DLBCL tumors, future nanocarrier designs must account for this heterogeneity, either through multi-ligand platforms capable of targeting multiple receptors simultaneously, or through combination strategies that pair receptor-targeted nanocarriers with agents targeting clonally ubiquitous resistance mechanisms such as BCL-2 or NF-κB pathway activation [89].

The immune landscape of individual DLBCL tumors represents a third axis of patient-specific variability with significant implications for nanocarrier efficacy. In a retrospective study of 174 DLBCL patients, the positive rates of tumor-infiltrating lymphocyte (TIL)-associated immune checkpoints were: PD-1 (79.3%), LAG-3 (78.8%), TIM-3 (62.7%), and TIGIT (69.5%), with high LAG-3 expression independently predicting inferior prognosis [90]. This immune checkpoint landscape is highly variable between patients, suggesting that the TME of an individual DLBCL patient—ranging from an immunologically “hot” tumor with abundant T-cell infiltration to a “cold” tumor with an immunosuppressive milieu—fundamentally determines whether nanocarrier-mediated drug delivery will encounter a permissive or hostile immune context. For nanocarrier strategies combining chemotherapy delivery with immune modulation, such as pH-sensitive carriers co-delivering checkpoint inhibitors with cytotoxic agents, patient stratification by PD-L1 expression, TIL density, or TME immune subtype is therefore not merely desirable but essential for identifying the responder population [91]. Emerging personalized approaches, such as loading patient-derived dendritic cell membranes onto nanocarrier surfaces to exploit tumor-specific antigen recognition, or engineering exosomes from autologous T cells to enable tumor homing, represent promising strategies to align nanocarrier design with the specific immune profile of each patient’s tumor [92]. Ultimately, the realization of truly personalized nanocarrier therapy in DLBCL will require the integration of COO profiling, comprehensive genomic characterization, and immune microenvironment assessment into a unified pre-treatment biomarker panel that guides both nanocarrier target selection and combination strategy—a vision that remains aspirational but increasingly within reach as multi-omics platforms become standard in clinical lymphoma practice.

Subcutaneous cell line xenografts—the predominant model in existing DLBCL nanocarrier literature—overestimate the EPR effect and fail to reproduce the disseminated nodal and bone marrow involvement of clinical disease. PDX models established in NSG mice preserve the histopathological and genetic features of donor tumors across passages, capture GCB/ABC subtype biology, and retain clinically acquired resistance mechanisms [93]. A publicly available DLBCL PDX repository of 48 molecularly characterized models now provides a standardized platform for comparative nanocarrier evaluation. For CXCR4- or TME-responsive nanocarriers specifically, disseminated intravenous engraftment models—in which lymphoma cells home to bone marrow and lymph nodes—are preferable to subcutaneous models for assessing biodistribution and penetration into sanctuary sites.

Conventional single-arm Phase I designs are insufficient to generate the biomarker-outcome data required to guide nanocarrier development. Biomarker-enriched designs restricting enrollment to target-receptor-positive patients (e.g., TFR1-high, CXCR4-high) offer the most efficient path to early efficacy signals [94]. Where multiple platforms are evaluated in parallel, umbrella trials screening patients for a biomarker panel and assigning them to matched nanocarrier arms enable simultaneous hypothesis testing within a single master protocol [95]. Window-of-opportunity trials—administering the nanocarrier between diagnosis and R-CHOP initiation with pre/post-treatment tumor sampling—can provide pharmacodynamic proof-of-concept including intratumoral drug concentration and target engagement in treatment-naive patients.

Clinical translation landscape: current status of nanocarrier-based therapies in DLBCL

Despite the promising preclinical advances described in previous sections, the clinical translation of nanocarrier-based therapies for DLBCL remains in its early stages. To date, the most clinically validated nanocarrier platform in DLBCL is liposomal doxorubicin (LD), which substitutes conventional doxorubicin in the R-CHOP backbone—giving rise to the R-COMP (non-pegylated LD) and R-CDOP (pegylated LD) regimens. Multiple clinical trials have evaluated these liposomal formulations, and their outcomes offer important translational lessons.

The Phase II EUR018 trial enrolled 72 elderly DLBCL patients treated with R-COMP, reporting an overall response rate (ORR) of 71% and a complete remission (CR) rate of 57%, with a 3-year overall survival of 72% [96]. These results demonstrated that liposomal doxorubicin could preserve antitumor efficacy while reducing anthracycline-associated cardiotoxicity in an elderly, comorbid population. This finding was further substantiated in the randomized Phase III AGMT NHL-14 trial, which directly compared R-COMP to standard R-CHOP. Although no significant difference in antitumor efficacy was observed, the incidence of severe LVEF decline below 50% was significantly lower in the R-COMP arm (4.6% vs. 15.8%, P < 0.001), confirming the cardioprotective potential of nanocarrier-encapsulated anthracyclines [97]. The multicenter HEART01 Phase II trial further validated R-COMP in DLBCL patients with pre-existing cardiac disorders, achieving an ORR of 72% with manageable cardiac toxicity [98]. More recently, a randomized Phase II study from the Spanish GELTAMO group (NCT02012088) corroborated these findings in patients aged ≥ 60 years, demonstrating comparable efficacy between R-COMP and R-CHOP, along with a significant reduction in troponin elevation (63% vs. 100% at cycle 6, P = 0.001), a sensitive biomarker of subclinical myocardial injury [99]. In parallel, a multi-center randomized Phase IV study from China reported that R-CDOP, incorporating pegylated LD, achieved superior CR/PR ratios compared to R-CHOP in newly diagnosed DLBCL patients (P < 0.05), while demonstrating more favorable cardiac enzyme profiles at 9 months post-treatment.

Taken together, these trials establish that liposomal doxorubicin formulations are non-inferior in efficacy and potentially superior in cardiac safety compared to conventional doxorubicin. However, a critical limitation must be acknowledged: these liposomal platforms function primarily as passive nanocarriers for drug delivery, and their clinical benefit is centered on toxicity reduction rather than the reversal of chemotherapy resistance. Crucially, none of the more sophisticated resistance-targeting nanocarrier strategies discussed in this review—including CXCR4-targeted nanoparticles, TFR1-directed carbon-nitride dots, siRNA-loaded exosomes, or stimuli-responsive systems—have advanced to clinical trials in DLBCL to date. This represents a profound translational gap between the preclinical innovation landscape and clinical reality. The key lessons learned from existing liposomal trials, including the heterogeneity of the EPR effect in hematological malignancies, the challenges of patient selection, and the need for biomarker-driven trial design, should inform the development of next-generation actively targeted nanocarrier trials. A comprehensive overview of the current clinical trial landscape is summarized in Table 3.

Table 3.

Clinical trials of nanocarrier-based therapies in DLBCL: current status, outcomes, and lessons learned

Trial/study	Phase	Nanocarrier type	Regimen	Patient population	Key outcomes	Lessons Learned	References
EUR018 trial	Phase II	Non-pegylated liposomal doxorubicin (NPLD)	R-COMP versus historical R-CHOP controls	Elderly DLBCL, n = 72, median age 72 yrs	ORR 71%; CR 57%; 3-yr OS 72%; Grade 3–4 neutropenia 54%	NPLD preserves efficacy in elderly/comorbid patients; passive nanocarrier delivery reduces peak anthracycline exposure but does not address drug resistance mechanisms	[96]
AGMT NHL−14	Phase III (randomized)	Non-pegylated liposomal doxorubicin (NPLD)	R-COMP versus R-CHOP	Untreated CD20 + DLBCL, n ≈ 200	No significant efficacy difference; LVEF < 50% incidence: R-COMP 4.6% vs. R-CHOP 15.8% (P < 0.001)	Cardioprotection confirmed in normal cardiac function patients; EPR-based passive targeting insufficient for resistance reversal; efficacy equivalence ceiling limits further development	[97]
HEART01 trial	Phase II	Non-pegylated liposomal doxorubicin (NPLD)	R-COMP	DLBCL with pre-existing cardiac disorders, n = 50, median age 76 yrs	ORR 72%; CR 56%; Grade 3–4 cardiac events in 6 patients; LVEF stable throughout	Liposomal formulation enables anthracycline delivery in otherwise ineligible cardiac patients; highlights unmet need for nanocarriers tailored to high-risk subpopulations	[98]
GELTAMO trial (NCT02012088)	Phase II (randomized)	Non-pegylated liposomal doxorubicin (NPLD)	R-COMP versus R-CHOP	DLBCL ≥ 60 yrs, LVEF ≥ 55%, n = 90	Efficacy equivalent; troponin elevation lower in R-COMP arm (63% vs. 100% at cycle 6, P = 0.001); Grade ≥ 3 cardiac events: R-CHOP 4 vs. R-COMP 0	Troponin as sensitive biomarker for subclinical cardiotoxicity; R-COMP does not improve survival but reduces cardiac risk signals; biomarker-guided patient selection warranted	[99]
Chinese multicenter Phase IV	Phase IV (randomized)	Pegylated liposomal doxorubicin (PLD)	R-CDOP versus R-CHOP	Newly diagnosed DLBCL/FL, n = 102	CR/PR ratio superior in R-CDOP group (P < 0.05); cardiac enzyme levels (CK, CKMB) significantly lower in R-CDOP at 9 months	PLD formulation offers both efficacy and cardiac safety advantages in younger patients; long-term data lacking; short follow-up (3–9 months) limits survival conclusions	[100]
Li et al. 2023	Retrospective cohort	Pegylated liposomal doxorubicin (PLD)	RCdOP/RCDOP versus RCEOP (epirubicin control)	Elderly DLBCL (60–85 yrs), n = 335	PLD and epirubicin showed similar OS (P = 0.776) and PFS (P = 0.959); full-dose RCDOP (30–45 mg/m²) showed higher CR rate vs. low-dose (75.9%, P = 0.018)	Optimal PLD dosing is critical; full-dose PLD preferred in patients with cardiovascular comorbidities; dose intensity directly impacts CR rates	[101]
CXCR4-targeted nanocarrier (T22-DITOX-H6)	Preclinical	Protein-based CXCR4-targeted nanoparticle	T22-DITOX-H6	CXCR4 + DLBCL mouse models	Tumor-selective uptake (70–80% of administered dose); antitumor effect without systemic toxicity in vivo	No clinical trial initiated; CXCR4 heterogeneity within tumors may limit response; scalable GMP production of protein nanocarriers remains a challenge	[1]
TFR1-targeted CND nanocarrier (CDT)	Preclinical	Carbon-nitride dot (CND)-Dox-Transferrin conjugate	CDT (CND-Dox-TF)	DLBCL PDX mouse models	10–100× greater potency vs. free Dox in vitro; improved OS in PDX models with reduced host toxicity	Patent filed; no clinical trial registered; TFR1 expression heterogeneity requires biomarker stratification; clinical translation pending	[28]
siRNA nanocarrier (exosome-PDK4)	Preclinical	Exosome-based siRNA nanoparticle	siPDK4 + anti-CD20	DLBCL cell lines and mouse models	Reversal of rituximab resistance via PDK4 silencing; restored CD20 expression and chemosensitivity	No clinical trial initiated; exosome batch consistency and scalability remain major barriers; off-target gene silencing risk requires rigorous safety evaluation	[35]

ORR, overall response rate; CR, complete remission; OS, overall survival; PFS, progression-free survival; LVEF, left ventricular ejection fraction; NPLD, non-pegylated liposomal doxorubicin; PLD, pegylated liposomal doxorubicin; PDX, patient-derived xenograft; GMP, good manufacturing practice. Preclinical entries are included to illustrate the translational gap between laboratory innovation and clinical application.

Regulatory landscape: pathways and requirements for clinical approval of nanocarrier-based therapies

The clinical translation of nanocarrier-based therapies is as much a regulatory challenge as a scientific one. Unlike conventional small-molecule drugs, nanocarriers are complex systems whose physicochemical properties directly govern their biological behavior, safety, and efficacy—properties that existing regulatory frameworks were not originally designed to evaluate. Neither the FDA nor the EMA has established a dedicated nanomedicine regulatory category, resulting in a landscape that is simultaneously evolving and fragmented across major jurisdictions [102].

The FDA’s 2022 guidance on drug products containing nanomaterials adopts a risk-based framework, requiring sponsors to define and control critical quality attributes (CQAs)—including particle size distribution, zeta potential, morphology, drug loading efficiency, and in vitro release kinetics—throughout the product lifecycle [FDA, 2022]. The EMA similarly defines nanomedicines as systems in the 1–1000 nm range with specific properties unachievable by macro-scale systems, and has issued reflection papers on liposomes, polymeric nanoparticles, and iron colloids [62]. A key structural advantage in the EU is the EMA’s Innovation Task Force (ITF), which offers early informal scientific consultations for complex nanomedicine products—nanotechnology has been among the top 10 ITF discussion themes from 2019 to 2022. Critically, neither the FDA’s ANDA nor the EMA’s hybrid application pathway adequately accommodates nanosimilars, as demonstrating bioequivalence at the nanoscale remains technically and legally unresolved [103].

Prior to an IND submission, nanocarrier developers must fulfill a substantially more extensive preclinical safety evaluation than for conventional drugs. A three-tiered hazard evaluation strategy—aligned with the standardized assay cascades of the NCI Nanotechnology Characterization Laboratory (NCI-NCL) and the European Nanomedicine Characterisation Laboratory (EU-NCL)—progresses from physicochemical characterization, through cellular uptake and intracellular persistence assessment, to tailored toxicological assays [104]. For DLBCL-targeted nanocarriers specifically, mandatory preclinical data should include immunogenicity profiling (complement activation and cytokine induction, given the heightened CRS risk in lymphoma patients), protein corona characterization, and in vivo pharmacokinetic and biodistribution studies in relevant models [18]. A persistent regulatory concern is that most DLBCL preclinical studies rely on subcutaneous xenograft models that overestimate the EPR effect relative to clinical reality; IND packages should ideally incorporate patient-derived xenograft (PDX) or disseminated lymphoma models to generate more regulatorily credible translational data [105].

For R/R DLBCL—a disease with significant unmet medical need—the FDA’s accelerated approval pathway offers an expedited route contingent on surrogate endpoint demonstration (e.g., overall response rate), with confirmatory post-marketing trials required [FDA Draft Guidance, 2023]. However, the FDA’s 2023 draft guidance on oncology accelerated approvals signals a shift toward randomized controlled trials over single-arm studies, meaningfully increasing the complexity and cost of nanomedicine clinical development programs. Nanocarrier-specific safety monitoring beyond standard oncology requirements must also be incorporated into trial protocols, including prospective assessment of infusion-related reactions, complement activation-related pseudoallergy (CARPA), and the accelerated blood clearance (ABC) phenomenon associated with anti-PEG antibody formation. The non-linear pharmacokinetics of nanocarriers further necessitate more intensive PK sampling strategies than conventional drugs to support dose selection across patient subgroups.

The absence of globally harmonized nanomedicine definitions, combined with divergent national requirements, contributes to approval timelines disproportionate to the data being submitted—approximately 30% of EMA nanomedicine applications fail due to inadequate manufacturing data alone [106]. To accelerate clinical translation, the field requires: adoption of Quality-by-Design (QbD) principles from the earliest development stages; early engagement with regulators via pre-IND meetings (FDA) or ITF consultations (EMA); and ICH-level international harmonization of nanocarrier-specific guidelines [107].

Conclusion and Future Considerations

The treatment of DLBCL using chemotherapy faces resistance that limits the effectiveness of treatment and patient outcomes. Various solutions are developed to resolve these challenges, including multifunctional nanocarriers, because these are able to control the resistance mechanism with co-delivery, gene therapy, and targeted delivery strategies. Further challenges are of safety, scalability, and biological barriers that need to be reduced for effective patient outcomes in DLBCL. The pre-clinical and clinical research can be managed in the future for validations and trials of nanocarriers that can further assist to improve treatment among DLBCL patients. The future integration is supposed to have multiple functionalities, including gene therapy, targeted delivery, and controlled chemotherapies release. The ultimate aim is to increase the DLBCL therapeutic outcomes. Furthermore, the adoption of a robust in vivo model is also required to assess the safety, efficacy, and pharmacokinetics of nanocarriers. The findings can be validated with clinical trials, and consistent testing is also needed for nanocarriers to attain regulatory approvals, increase efficacy, and minimize toxicity.

In the context of DLBCL, the clinical application of multifunctional nanocarriers—particularly those incorporating active targeting ligands or biomimetic membranes—must account for the risk of Cytokine Release Syndrome (CRS). Lymphoma patients often present with a “primed” immune environment characterized by systemic dysregulation and high tumor burden, significantly lowering the threshold for massive cytokine release. Nanoparticle-induced CRS typically stems from the non-specific activation of monocytes and macrophages during systemic circulation. Furthermore, the dose-dependent nature of nanocarrier-induced immune activation necessitates rigorous dose-escalation protocols and potentially the prophylactic use of IL-6 receptor antagonists, such as tocilizumab, to mitigate systemic inflammatory cascades [108].

Author contributions

DL was a major contributor in writing the manuscript. KH and XK were the main supervisor and reviewed the manuscript. All authors read and approved the final manuscript.

Funding

No funding.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.