Prospects and Challenges of Different Delivery Systems in Breast Tumors Therapy

Characterisation of HA

Hyaluronic acid enhances drug uptake in cells with high expression of its receptors, thereby potentiating therapeutic efficacy. Cluster of differentiation 44, the primary receptor for HA, is a transmembrane glycoprotein that exhibits elevated expression in various tumors. The CD44 receptor undergoes alternative exon splicing and posttranscriptional modifications in gene expression, resulting in multiple isoforms. ⁸⁰ Anttila et al ⁸¹ demonstrated that stromal HA accumulation correlates with tumor progression by evaluating HA levels and CD44 expression; however, they argued that HA accumulation is not associated with CD44 expression on the surface of human ovarian cancer cells. Zhao et al ⁸² found that CD44 promotes cell proliferation in lung cancer cells through activation of mitogen-activated protein kinase (MAPK) signaling, whereas deletion of the CD44 gene inhibits Kras-mediated lung adenocarcinoma proliferation in mice. Furthermore, the interaction between HA and CD44 facilitates ankyrin binding to MDR-1, leading to the efflux of chemotherapeutic drugs and chemotherapy resistance in tumor cells. ⁸³

Targeted therapy with HA

Hyaluronic acid demonstrates excellent biocompatibility and exhibits no immunogenicity. ⁸⁴ HA can bind to the overexpressed cluster of differentiation protein 44 (CD44) receptors on cancer cell membranes, thereby conferring tumor-targeting properties. Notably, CD44 receptors are upregulated in various BC subtypes, with significant overexpression observed in TNBC. Li et al ⁸⁵ synthesized targeted micelles with an average diameter of 120 nm using an HA-deoxycholic acid (DOCA) copolymer loaded with paclitaxel (Taxol), achieving a drug loading content of 34%. MTT assays were performed to analyze and compare the cytotoxicity of this formulation with clinically used paclitaxel in MDA-MB-231 BC cells and normal human embryonic lung fibroblasts (HELF). The results indicated that paclitaxel-loaded HA micelles exhibited enhanced cytotoxicity against CD44-positive tumor cells compared with paclitaxel alone, while demonstrating reduced toxicity toward HELF cells at equivalent concentrations. Studies have highlighted that breast tumor cells contain high levels of hyaluronidase (HAase), which can rapidly degrade HA-based drug carriers, releasing the drug and targeting tumor cells.^86,87 Stenzel ⁸⁸ developed a pH-responsive HA micellar system by conjugating thiolated HA (HA-SH) with the lateral unsaturated bonds of phosphatidylcholine via click chemistry methodology. The resultant copolymer spontaneously formed doxorubicin-encapsulated micelles, achieving a 10% drug loading efficiency. The release kinetics were evaluated at pH 7.4 and 5.0, revealing approximately 2-fold higher doxorubicin release in an acidic environment (pH 5.0) compared with physiological conditions (pH 7.4) over a 48-h period. Wang et al ⁸⁹ developed HA-modified liposomes loaded with cisplatin (CDDP) and hesperidin (Hes), enhancing the efficacy of chemotherapy in TNBC. Hyaluronic acid modification promotes the uptake and retention of nanoparticles in BC cell lines. These nanoparticles mitigate inflammation by inhibiting the PI3K/Akt/mTOR pathway and improve chemosensitivity by inhibiting epithelial-mesenchymal transition, effectively suppressing the invasion and metastasis of TNBC while exhibiting reduced side effects on normal tissues.

Hydrogen sulfide (H2S) has been recognized as the third gasotransmitter implicated in various physiological and pathological processes. Endogenous H2S or moderate exogenous H2S levels promote tumor cell survival, whereas increased expression of H2S-generating enzymes or exposure to high concentrations of H2S-releasing compounds may exhibit antitumor effects.^90,91 ADT-OH, a commonly used H2S-releasing agent, contains the ~3H-1,2-dithiol-3-thione moiety within its molecular structure. ⁹² The synthesis of HA-ADT is achieved through the chemical conjugation of HA with ADT-OH. Dong et al ⁹³ investigated the effects of HA-ADT on human BC cell progression. The findings revealed that HA-ADT released higher levels of H2S compared with NaHS and GYY4137. Hyaluronic acid-ADT exhibited superior inhibitory effects on the proliferation and viability of human BC cells relative to NaHS and GYY4137, while also reducing their migratory and invasive capabilities. These results suggest that HA-ADT may play a pivotal role in suppressing the growth, migration, and invasion of human BC cells. The integration of these in vitro findings supports the development of a promising formulation with enhanced tumor-targeting efficacy and improved safety for normal cells, highlighting the potential benefits of active tumor targeting. ⁹⁴

As evident from the above, both in vitro and in vivo studies robustly support the concept that conjugating HA to the outer surface of micelles enhances the targeting of tumor cells. When combined with passive targeting, this active targeting strategy offers a promising approach for delivering chemotherapeutics to tumors that overexpress CD44. ⁹⁵ As depicted in Figure 5, HA-based nanomicelles exhibit excellent biological safety and possess notable capabilities for drug release, blood compatibility, and systemic tumor targeting, thus presenting a favorable prospect for clinical oncology treatment.^96,97

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Figure 5.

Structure and function of hyaluronic acid (HA).

Characteristics of US microbubble for drug delivery

The efficacy of chemotherapy for tumors depends on the effective concentration and duration of chemotherapeutic drugs at the tumor site. Chemotherapeutic agents can prolong survival in BC patients; however, adverse reactions remain the primary issue limiting their clinical application. Paclitaxel, a lipophilic drug, is widely used as a chemotherapeutic agent for BC but often induces numerous adverse reactions during chemotherapy. Encapsulating paclitaxel within microbubbles can reduce drug toxicity and side effects while enhancing local drug concentration within the tumor. Tang et al ¹⁰⁰ investigated the effects of UTMD-mediated suppressor of cytokine signaling 3 (SOCS3) gene therapy on the biological characteristics and epithelial-mesenchymal transition of BC stem cells by establishing an in vivo BC model and culturing BC stem cells in vitro. The results indicated that UTMD-mediated SOCS3 gene therapy exhibited superior therapeutic effects. Takabatake et al ¹⁰¹ studied the inhibitory impact of gefitinib and taxane microbubble complexes on BC cell lines MDA-MB-231 (EGFR-positive) and MCF7/ADR (EGFR and HER2-positive). These findings suggested that gefitinib and taxane microbubble complexes could overcome drug resistance in these cancer cells, thereby enhancing the effect of taxanes on MCF7/ADR cells.

Researchers have also prepared dual-modified lipid CLR8LP (DOX + VER) microbubbles co-loaded with doxorubicin (DOX) and verapamil (VER) to investigate their inhibitory effects on the proliferation of human BC MCF7 cells. The results showed that co-encapsulating DOX and VER within co-modified liposomes significantly increased the cytotoxic efficacy against MCF7 cells. Overexpression of P-glycoprotein (Pgp) on tumor cell membranes is one of the primary mechanisms of drug resistance.^{102 -104} A commonly used strategy is to combine Pgp inhibitors with chemotherapeutic drugs. Verapamil, as a Pgp inhibitor, and dual-modified liposomes (CLR8LP) with excellent tumor-targeting delivery capabilities were used as carriers for chemotherapeutic drugs and Pgp inhibitors to create microbubble complexes. This enables chemotherapeutic drugs, with the synergistic effect of Pgp inhibitors, to effectively accumulate within cells, inhibiting and killing tumor cells. Ultrasound microbubbles carrying drugs not only reduce drug toxicity and side effects but also increase local drug concentration within tumors, enhancing the cytotoxic effect on tumor tissues.

Targeting of US microbubble in therapy

Ultrasonic microbubbles, as carriers, can not only reduce drug side effects in systemic circulation while enhancing drug concentration in pathological regions but also protect drugs and gene fragments from degradation by nucleases and clearance by the reticuloendothelial system. Gene therapy involves the use of gene engineering techniques to introduce exogenous genes into target cells for correcting functional defects or providing new functions. ¹⁰⁵ Ultrasonic targeted microbubble destruction technology represents a targeted and efficient gene delivery method that minimizes injury, as supported by the excellent cavitation effect of microbubbles. ¹⁰⁶ Some researchers ¹⁰⁷ combined targeted ultrasonic microbubbles with CD/TK bicistronic suicide gene plasmids and prodrugs to transfect BC MCF7 cells expressing the KDR promoter, followed by US irradiation. The results demonstrated that the KDR promoter could drive the targeted expression of CD/TK bicistronic suicide genes in MCF7 cells, and the KDRP-CD/TK gene exhibited a targeted cytotoxic effect on MCF7 cells in vitro. Survivin, a member of the inhibitor of apoptosis protein family, promotes tumor proliferation. By inhibiting survivin gene expression and interfering with survivin Thr34 phosphorylation, cancer cells undergoing mitosis can be induced to undergo apoptosis, thereby enhancing the apoptosis-inducing effects of chemotherapeutic drugs on cancer cells. ¹⁰⁸ Researchers have constructed an siRNA eukaryotic expression vector to silence the human survivin gene ¹⁰⁹ and prepared lipid microbubbles loaded with this vector using the reverse-phase evaporation method with mechanical agitation. In vitro experiments showed that gene-loaded targeted microbubbles could specifically bind to BC cells with high HER-2 expression and induce apoptosis. The safety, efficacy, and stability of microbubbles as gene carriers have been confirmed in numerous experiments conducted by scholars worldwide; however, their clinical application still faces many challenges. Some scholars ¹¹⁰ noted as early as the 2015 Annual Meeting of the American Association for Cancer Research (AACR) that MPDL3280A exhibited durable efficacy in advanced TNBC. Programmed death 1/PDL1 immunotherapy is currently a hot topic in antitumor therapy, but no studies have been reported on conjugating anti-PD1 monoclonal antibodies to microbubbles for BC treatment.

This indicates that ultrasonic microbubbles can enhance the permeability of tumor cell membranes, induce mechanical damage to vascular endothelium and adjacent tumor tissues, and enlarge endothelial gaps. Ultrasonic microbubbles are capable of delivering chemotherapeutic agents such as paclitaxel in a targeted manner, thereby reducing drug toxicity and side effects, increasing local drug concentration in tumors, and potentiating the cytotoxic effects of chemotherapeutics on tumor cells. Ultrasonic microbubbles can facilitate the introduction of exogenous genes into BC cells to correct functional defects in target cells and promote apoptosis. Furthermore, they can deliver monoclonal antibody carriers for targeted immunotherapy, activating the immune system, enhancing immune responses, and inhibiting tumor initiation and progression. Currently, there are limited reports on microbubbles carrying drugs and genes targeting tumor parenchymal cells. Therefore, future research should focus on identifying safe and efficient targets for tumor parenchymal cells to improve the precision of ultrasonic microbubble-mediated targeted therapy.

The mechanism of action of ADCs

The design of ADCs comprises 3 critical components: the antibody, the cytotoxic drug, and the linker. ¹¹² Antibody-drug conjugates combine the highly specific targeting capability of antibodies with potent small-molecule cytotoxic agents, maintaining stability in the circulatory system. As depicted in Figure 6, upon binding to antigens expressed on the surface of tumor cells, ADCs undergo intracellular degradation and release their payload within the cell, achieving a high local drug concentration that effectively and precisely eliminates tumor cells. By integrating the targeting properties of monoclonal antibodies with the cytotoxicity of chemotherapeutic agents, ADCs represent a significant advancement over traditional chemotherapy and targeted therapy. ¹¹³ When specific antigens on the surface of tumor cells are kinase receptors or molecules involved in signaling pathways associated with tumor growth, differentiation, and proliferation, the antibody component of ADCs binds to these antigens, thereby blocking downstream signaling pathways and inhibiting tumor cell proliferation. ¹¹⁴ In addition, ADCs can induce apoptosis via antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). ¹¹⁵

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Figure 6.

Hyaluronic acid-decorated nanomicelle-mediated active and passive targeting.

Trastuzumab emtansine (T-DM1) is a novel ADC that demonstrates biological activity analogous to trastuzumab. It selectively targets and delivers the potent antimicrotubule agent DM-1 to HER-2-positive tumor cells, effectively and specifically eliminating them. The initial success of T-DM1 in BC treatment has expedited advancements in this field, with over 60 new ADCs currently in development.^{116 -118} Nevertheless, ADC development continues to encounter substantial challenges, including complex drug structures, targeting efficiency, and adverse effects, which have contributed to suboptimal outcomes in clinical trials. ¹¹⁹ Of particular significance is the balance between lower-than-expected efficacy and higher-than-expected toxicity, highlighting the necessity for further innovation in designing next-generation ADCs. ¹²⁰ In summary, ADCs provide novel opportunities to enhance the prognosis of BC patients. Through ongoing optimization of drug molecule design and target selection, improved treatment options for BC can be anticipated in the future. ¹²¹

Clinical application of ADCs

Antibody-drug conjugates selectively deliver cytotoxic agents or radioisotopes to malignant cells through targeted monoclonal antibodies. ¹²² In the TNBC field, ADCs that have entered clinical trials include DS-8201a, sacituzumab govitecan, ladiratuzumab vedotin (SGN-LIV1A), CAB-ROR2-ADC, anti-CA6-DM4 immunoconjugate (SAR566658), AVID100, and U3-1402. ¹²³ Although atezolizumab and albumin-bound paclitaxel have been approved, their combination is currently limited to PD-L1-positive TNBC patients, highlighting ADCs as a promising targeted therapeutic option for the remaining 60% of PD-L1-negative patients.

DS-8201, also known as T-DXd, is an ADC comprising trastuzumab, a peptide-based cleavable linker, and a topoisomerase I inhibitor payload. ¹²⁴ The amino acid sequence of its antibody component is identical to that of trastuzumab, and it is conjugated to the cytotoxic agent DXd via a protease-cleavable tetrapeptide linker, achieving a drug-to-antibody ratio (DAR) of 8. The phase III DESTINY-Breast03 trial demonstrated that T-DXd significantly reduced the risk of mortality by 36% in patients with HER2-positive BC, with a median progression-free survival (PFS) more than 4 times longer compared with the T-DM1 arm (28.8 months vs. 6.8 months, HR = 0.33, P < 0.0001). In addition, 79% of participants achieved an objective response, including 21% who attained a complete response. ¹²⁵ These findings indicate that, compared with T-DM1, T-DXd provides clinically and statistically significant improvements in survival and sustained PFS benefits for patients with pretreated HER2-positive metastatic BC. Furthermore, T-DXd has shown manageable and tolerable safety profiles even with prolonged treatment exposure. Recently, results from a phase I dose-escalation study (NCT02564900) were published to evaluate the safety and tolerability of DS-8201 in HER2-positive BC patients who progressed after prior T-DM1 therapy. ¹²⁶ The dosing regimens were 5.4 mg/kg or 6.4 mg/kg every 3 weeks, and common grade 3 or higher adverse events included anemia (17%), neutropenia (14%), leukopenia (9%), and thrombocytopenia (8%). The trial confirmed controllable safety for DS-8201. Moreover, an ongoing phase III clinical trial (NCT03529110) is enrolling 500 patients with HER2-positive, unresectable, and/or metastatic BC who have received prior trastuzumab or taxane-based chemotherapy. This trial compares DS-8201 to T-DM1, with PFS as the primary endpoint and overall survival (OS) and objective response rate (ORR) as secondary endpoints.

Trophoblast cell surface antigen 2 (Trop-2), a glycoprotein widely expressed in various tumor types, is also referred to as tumor-associated calcium signal transducer 2 (TACSTD2), membrane component 1 surface marker 1 (M1 S1), gastrointestinal antigen 733-1 (GA733-1), and epithelial glycoprotein 1 (EGP-1). First identified in a human choriocarcinoma cell line in 1981, it belongs to the category of cell surface receptors. Trop-2 serves as a critical signaling molecule regulating tumor growth. ¹²⁷ It is overexpressed in malignant tumors such as BC, gastric cancer, colorectal cancer, and nonsmall cell lung cancer. Through mediation of multiple signaling pathways, Trop-2 regulates cell proliferation and apoptosis, thereby participating in tumor development. ¹²⁸ Particularly in TNBC cells, this characteristic makes Trop-2 an ideal target for ADCs. ¹²⁹ Sacituzumab govitecan (SG), a Trop-2-targeted ADC, exerts antitumor activity by conjugating an anti-Trop-2 antibody with the cytotoxic agent SN-38 (7-ethyl-10-hydroxycamptothecin). ¹³⁰ The final results of the phase III ASCENT study demonstrated that SG significantly prolonged median PFS (5.6 months vs. 1.7 months, HR = 0.39, P < 0.001) and overall survival (OS) (12.1 months vs. 6.7 months, HR = 0.48, P < 0.001) compared with single-agent chemotherapy in patients with unresectable, recurrent, or refractory locally advanced/metastatic TNBC who had received ⩾ 2 prior lines of standard chemotherapy. The most common grade 3/4 treatment-related adverse events were neutropenia and diarrhea, which could be managed with symptomatic support. Furthermore, the final analysis of the phase III TROPiCS-02 study indicated that SG was superior to physician’s choice of chemotherapy in patients with endocrine therapy-resistant metastatic hormone receptor (HR)-positive/HER2-negative BC who had previously received taxanes, endocrine therapy, CDK4/6 inhibitors, and 2-4 lines of chemotherapy, with median OS of 14.4 months versus 11.2 months, respectively (HR = 0.79, P = 0.02). ¹³¹ A single-arm, multicenter, phase II clinical trial evaluated the efficacy and safety of sacituzumab govitecan in patients with recurrent/refractory metastatic TNBC. ¹³² Results showed an objective response rate (ORR) of 30%, including 2 complete responses and 19 partial responses, with a median PFS of 6 months and median OS of 16.6 months. Immunohistochemical analysis revealed moderate to strong Trop-2 expression in 88% of patients. The most common grade ⩾ 3 adverse events included neutropenia (39%), leukopenia (16%), anemia (14%), and diarrhea (13%), with a febrile neutropenia incidence rate of approximately 7%. The trial confirmed the favorable efficacy and tolerability of sacituzumab govitecan in metastatic TNBC. Another ongoing multicenter, randomized, open-label, phase III clinical trial (NCT02574455) is enrolling patients with refractory/metastatic TNBC or recurrence after at least 2 prior lines of chemotherapy. This trial compares sacituzumab govitecan to the physician’s choice of chemotherapy (capecitabine, eribulin, gemcitabine, and vinorelbine), with PFS as the primary endpoint and OS, ORR, and other indicators as secondary endpoints.

SGN-LIV1A represents an additional ADC therapeutic, comprising a monoclonal antibody targeting LIV1 conjugated to the microtubule inhibitor monomethyl auristatin E (MMAE). ¹³³ LIV1 is a transmembrane protein with zinc transporter and metalloproteinase activities, exhibiting high expression in over 90% of breast tumors but limited expression in normal tissues. ¹³⁴ An ongoing open-label, dose-escalation Phase I clinical trial (NCT01969643) is evaluating the safety and tolerability of SGN-LIV1A in patients with metastatic BC. The interventional strategies in this study include the administration of SGN-LIV1A as a single agent or in combination with trastuzumab. The primary endpoint is the incidence of adverse events, while secondary endpoints include blood concentrations of SGN-LIV1A and its metabolites, overall response rate (ORR), and other indicators. Currently, multiple clinical studies for this drug are underway, with results anticipated within the next 2 years. Other ADCs under investigation include CAB-ROR2-ADC, SAR566658, AVID100, and U3-1402. CAB-ROR2-ADC integrates conditionally active biologics (utilizing BioAtla’s proprietary CAB technology) with an antibody targeting ROR2, and its tolerability, safety, and antitumor activity are being assessed in patients with solid tumors. The anti-CA6-DM4 immunoconjugate (SAR566658) is an ADC consisting of a ds6 antibody targeting carbonic anhydrase 6 (CA6) coupled with the microtubule inhibitor DM4. ¹³⁵ AVID100 is the first epidermal growth factor receptor (EGFR)-targeted ADC to achieve clinical progress. AVID100 was evaluated in a multicenter, dose-escalation Phase IIa clinical trial in TNBC patients, initially demonstrating promising results, ¹³⁶ but recent updates indicate the trial was terminated due to insufficient efficacy. U3-1402 is an ADC where the HER3 monoclonal antibody patritumab is conjugated to the topoisomerase I inhibitor deruxtecan (Dxd) via a tetrapeptide linker. ¹³⁷ Previous data indicate that HER3 is expressed in approximately 50% to 70% of BC patients, yet few therapies specifically target HER3. ¹³⁸ At the 2022 ASCO meeting, the novel HER3-targeted ADC patritumab deruxtecan demonstrated efficacy in treating HER3-expressing metastatic BC. The U31402-A-J101 study enrolled 182 patients with HER3-expressing metastatic BC, including 113 HR +/HER2-, 53 TNBC, and 14 HER2+ patients. Following treatment with patritumab deruxtecan, overall survival (OS) was 14.6, 14.6, and 19.5 months, respectively, while PFS was 5.5, 7.4, and 11.0 months across the 3 subtypes. The study demonstrated the efficacy of patritumab deruxtecan irrespective of HER3 expression levels, with favorable safety, as only 10% of patients discontinued treatment due to intolerance. Researchers believe that HER3 will emerge as a critical target for treating diseases such as BC.

In summary, the application of ADCs in BC treatment has demonstrated significant progress and evolution in recent years. By leveraging the targeting specificity of antibodies to deliver cytotoxic agents to designated lesions, ADCs exert enhanced antitumor efficacy. Despite over 2 decades of investigation in oncology, ADC development has been relatively slow due to challenges in target selection, conjugation technology, antibody optimization, and drug release. However, advancements in fourth-generation ADC technology have established a promising contemporary landscape. In TNBC, SGN-LIV1A underwent a series of clinical trials, leading to the full FDA approval of the targeted anticancer agent Trodelvy, expanding its indication to adult patients who have received at least 2 prior systemic therapies, including 1 for metastatic or unresectable locally advanced TNBC. In addition, as depicted in Figure 7, ADCs such as ladiratuzumab vedotin, SAR566658, CAB-ROR2-ADC, and U3-1402 are currently under clinical evaluation (Figures 8, 9). ¹³⁹

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Figure 7.

Schematic of the microbubble-based, ultrasound-assisted photodynamic therapy (PDT) strategy. ⁹⁰ PDT, photodynamic therapy; PGL, porphyrin-grafted lipid; MB, microbubbles; LFUS, low-frequency ultrasound; CEUS, contrast-enhanced ultrasound.

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Figure 8.

Schematic representation of ADC drug structure and its mechanism of action.

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Figure 9.

Timeline of current and future ADC development in BC.