The Role of Checkpoint Inhibitor Expression Directly on Exfoliated Cells from Bladder Cancer: A Narrative Review.

Bladder cancer (BCa) is a common type of cancer that affects the urinary bladder. The early detection and management of BCa is critical for successful treatment and patient outcomes. In recent years, researchers have been exploring the use of biomarkers as a non-invasive and effective tool for the detection and monitoring of BCa. One such biomarker is programmed death-ligand 1 (PD-L1), which is expressed on the surface of cancer cells and plays a crucial role in the evasion of the immune system. Studies have shown that the PD-L1 expression is higher in BCa tumors than in healthy bladder tissue. Additionally, PD-L1 expression might even be detected in urine samples in BCa patients, in addition to the examination of a histological sample. The technique is being standardized and optimized. We reported how BCa patients had higher urinary PD-L1 levels than controls by considering BCa tumors expressing PD-L1 in the tissue specimen. The expression of PD-L1 in urinary BCa cells might represent both a diagnostic and a prognostic tool, with the perspective that the PD-L1 expression of exfoliate urinary cells might reveal and anticipate eventual BCa recurrence or progression. Further prospective and longitudinal studies are needed to assess the expression of PD-L1 as a biomarker for the monitoring of BCa patients. The use of PD-L1 as a biomarker for the detection and monitoring of BCa has the potential to significantly improve patient outcomes by allowing for earlier detection and more effective management of the disease.

p53/TP53 Status Assessment in Gastroesophageal Adenocarcinoma.

Chromosomal instability (CIN) is very frequent in gastroesophageal adenocarcinoma (GEA) and it is characterized by TP53 deletions/mutations resulting in p53 nuclear accumulation, as revealed by immunohistochemistry (IHC), which considers the cases with "high" staining levels to be positive. Aiming to improve aberrant TP53 detection, droplet digital PCR (ddPCR) was used to evaluate TP53 deletion in formalin-fixed, paraffin-embedded DNA (FFPE-DNA) and cell-free DNA (cfDNA). To further investigate the mutational TP53 profile, next-generation sequencing (NGS) was performed in a subset of FFPE samples. After combining "low" and "high" IHC staining level groups, the proportion of deletion events was significantly higher compared to the "intermediate" group (72.9% vs. 47.5%, p-value = 0.002). The ddPCR TP53 deletion assay was feasible for cfDNA but only had good agreement (72.7%, Cohen's kappa = 0.48) with the assay performed with FFPE-DNA of the "low-level" group. NGS analysis confirmed that, in the "low-level" group, a high percentage (66.7%) of cases were aberrant, with disruptive mutations that probably led to p53 loss. Data suggested that p53 IHC alone underestimates the CIN phenotype in GEA and that molecular analysis in both solid and liquid biopsies could be integrated with it; in particular, in cases of completely negative staining.

Putative Clinical Potential of ERBB2 Amplification Assessment by ddPCR in FFPE-DNA and cfDNA of Gastroesophageal Adenocarcinoma Patients.

Anti-HER2 monoclonal antibody trastuzumab improves the survival of those patients with advanced gastroesophageal adenocarcinoma (GEA) exhibiting HER2/ERBB2 overexpression/amplification. The current gold standard methods used to diagnose the HER2 status in GEA are immunohistochemistry (IHC) and silver or fluorescence in situ hybridization (SISH or FISH). However, they do not permit spatial and temporal tumor monitoring, nor do they overcome intra-cancer heterogeneity. Droplet digital PCR (ddPCR) was used to implement the assessment of HER2 status in formalin-fixed paraffin-embedded (FFPE) tumor DNA from a retrospective cohort (86 patients) and in cell-free DNA (cfDNA) samples from a prospective cohort (28 patients). In comparison to IHC/SISH, ddPCR assay revealed ERBB2 amplification in a larger patient fraction, including HER2 2+ and 0-1+ of the retrospective cohort (45.3% vs. 15.1%). In addition, a considerable number of HER2 2+ and 0-1+ prospective patients who were negative in FFPE by both IHC/SISH and ddPCR, showed ERBB2 amplification in the cfDNA collected just before surgery. cfDNA analysis in a few longitudinal cases revealed an increasing ERBB2 trend at progression. In conclusion, ddPCR in liquid biopsy may improve the detection rate of HER2 positive patients, preventing those patients who could benefit from targeted therapy from being incorrectly excluded.

PSMA-Specific CAR-Engineered T Cells for Prostate Cancer: CD28 Outperforms Combined CD28-4-1BB "Super-Stimulation".

Prostate cancer (PCa) is the second leading cause of malignancy-related mortality in males in the Western world. Although treatment like prostatectomy and radiotherapy for localized cancer have good results, similar positive outcomes are not achieved in metastatic PCa. Consequently, these aggressive and metastatic forms of PCa urgently need new methods of treatment. We already described an efficient and specific second-generation (2G) Chimeric Antigen Receptor (CAR) against Prostate Specific Membrane Antigen (PSMA), a glycoprotein overexpressed in prostate cancer and also present on neovasculature of several tumor entities. In an attempt to improve efficacy and in vivo survival of anti-PSMA 2G CAR-T cells, we developed a third generation (3G) CAR containing two costimulatory elements, namely CD28 and 4-1BB co-signaling domains, in addition to CD3ζ. Differently from what described for other 3G receptors, our third generation CAR disclosed an antitumor activity in vitro similar to the related 2G CAR that comprises the CD28 co-signaling domain only. Moreover, the additional costimulatory domain produced detrimental effects, which could be attributed to an increased activation-induced cell death (AICD). Indeed, such "superstimulation" resulted in an exhausted phenotype of CAR-T cells, after prolonged in vitro restimulation, a higher frequency of cell death, and an impairment in yielding sufficient numbers of transgenic T lymphocytes. Thus, the optimal combination of costimulatory domains for CAR development should be assessed cautiously and evaluated case-by-case.

Hyaluronan is a natural and effective immunological adjuvant for protein-based vaccines.

One of the main goals of vaccine research is the development of adjuvants that can enhance immune responses and are both safe and biocompatible. We explored the application of the natural polymer hyaluronan (HA) as a promising immunological adjuvant for protein-based vaccines. Chemical conjugation of HA to antigens strongly increased their immunogenicity, reduced booster requirements, and allowed antigen dose sparing. HA-based bioconjugates stimulated robust and long-lasting humoral responses without the addition of other immunostimulatory compounds and proved highly efficient when compared to other adjuvants. Due to its intrinsic biocompatibility, HA allowed the exploitation of different injection routes and did not induce inflammation at the inoculation site. This polymer promoted rapid translocation of the antigen to draining lymph nodes, thus facilitating encounters with antigen-presenting cells. Overall, HA can be regarded as an effective and biocompatible adjuvant to be exploited for the design of a wide variety of vaccines.

Anti-PSMA CAR-engineered NK-92 Cells: An Off-the-shelf Cell Therapy for Prostate Cancer.

Prostate cancer (PCa) has become the most common cancer among males in Europe and the USA. Adoptive immunotherapy appears a promising strategy to control the advanced stages of the disease by specifically targeting the tumor, in particular through chimeric antigen receptor T (CAR-T) cell therapy. Despite the advancements of CAR-T technology in the treatment of hematological malignancies, solid tumors still represent a challenge. To overcome current limits, other cellular effectors than T lymphocytes are under study as possible candidates for CAR-engineered cancer immunotherapy. A novel approach involves the NK-92 cell line, which mediates strong cytotoxic responses against a variety of tumor cells but has no effect on non-malignant healthy counterparts. Here, we report a novel therapeutic approach against PCa based on engineering of NK-92 cells with a CAR recognizing the human prostate-specific membrane antigen (PSMA), which is overexpressed in prostatic neoplastic cells. More importantly, the potential utility of NK-92/CAR cells to treat PCa has not yet been explored. Upon CAR transduction, NK-92/CAR cells acquired high and specific lytic activity against PSMA-expressing prostate cancer cells in vitro, and also underwent degranulation and produced high levels of IFN-γ in response to antigen recognition. Lethal irradiation of the effectors, a safety measure requested for the clinical application of retargeted NK-92 cells, fully abrogated replication but did not impact on phenotype and short-term functionality. PSMA-specific recognition and antitumor activity were retained in vivo, as adoptive transfer of irradiated NK-92/CAR cells in prostate cancer-bearing mice restrained tumor growth and improved survival. Anti-PSMA CAR-modified NK-92 cells represent a universal, off-the-shelf, renewable, and cost-effective product endowed with relevant potentialities as a therapeutic approach for PCa immunotherapy.

Real-Time Label-Free Targeting Assessment and in Vitro Characterization of Curcumin-Loaded Poly-lactic-co-glycolic Acid Nanoparticles for Oral Colon Targeting.

The exploitation of curcumin for oral disease treatment is limited by its low solubility, poor bioavailability, and low stability. Surface-functionalized poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) have shown promising results to ameliorate selective delivery of drugs to the gastro-intestinal tract. In this study, curcumin-loaded PLGA NPs (C-PLGA NPs) of about 200 nm were surface-coated with chitosan (CS) for gastro-intestinal mucosa adhesion, wheat germ agglutinin (WGA) for colon targeting or GE11 peptide for tumor colon targeting. Spectrometric and zeta potential analyses confirmed the successful functionalization of the C-PLGA NPs. Real-time label-free assessment of the cell membrane-NP interactions and NP cell uptake were performed by quartz crystal microbalance coupled with supported lipid bilayers and by surface plasmon resonance coupled with living cells. The study showed that CS-coated C-PLGA NPs interact with cells by the electrostatic mechanism, while both WGA- and GE11-coated C-PLGA NPs interact and are taken up by cells by specific active mechanisms. In vitro cell uptake studies corroborated the real-time label-free assessment by yielding a curcumin cell uptake of 7.3 ± 0.3, 13.5 ± 1.0, 27.3 ± 4.9, and 26.0 ± 1.3 µg per 104 HT-29 cells for noncoated, CS-, WGA-, and GE11-coated C-PLGA NPs, respectively. Finally, preliminary in vivo studies showed that the WGA-coated C-PLGA NPs efficiently accumulate in the colon after oral administration to healthy Balb/c mice. In summary, the WGA- and GE11-coated C-PLGA NPs displayed high potential for application as active targeted carriers for anticancer drug delivery to the colon.

A non-covalent antibody complex for the delivery of anti-cancer drugs.

Antibody drug conjugates (ADCs), which are obtained by coupling a potent cytotoxic agent to a monoclonal antibody (mAb), are traditionally bound in a random way to lysine or cysteine residues, with the final product's heterogeneity having an important impact on their activity, characterization, and manufacturing. A new antibody drug delivery system (ADS) based on a non-covalent linkage between a Fc-binding protein, in this case Protein A or Protein G, and a mAb was investigated in the effort to achieve greater homogeneity and to create a versatile and adaptable drug delivery system. Recombinant staphylococcal Protein A and streptococcal Protein G were chemically PEGylated at the N-terminus with a 5 kDa and a 20 kDa PEG, respectively, yielding two monoconjugates with a mass of ≈50 and ≈45 kDa. Circular dichroism studies showed that both conjugates preserved secondary structures of the protein, and isothermal titration calorimetry experiments demonstrated that their affinity for mAb was approximately 107 M-1. Upon complexation with a mAb (Trastuzumab or Rituximab), in vitro flow-cytometry analysis of the new ADSs showed high selectivity for the specific antigen expressing cells. In addition, the ADS complex based on Trastuzumab and Protein G, conjugated with a heterobifunctional 20 kDa PEG carrying the toxin Tubulysin A, had a marked cytotoxic effect on the cancer cell line overexpressing the HER2/neu receptor, thus supporting its application in cancer therapy.

Cell-autonomous and cell non-autonomous downregulation of tumor suppressor DAB2IP by microRNA-149-3p promotes aggressiveness of cancer cells.

The tumor suppressor DAB2IP contributes to modulate the network of information established between cancer cells and tumor microenvironment. Epigenetic and post-transcriptional inactivation of this protein is commonly observed in multiple human malignancies, and can potentially favor progression of tumors driven by a variety of genetic mutations. Performing a high-throughput screening of a large collection of human microRNA mimics, we identified miR-149-3p as a negative post-transcriptional modulator of DAB2IP. By efficiently downregulating DAB2IP, this miRNA enhances cancer cell motility and invasiveness, facilitating activation of NF-kB signaling and promoting expression of pro-inflammatory and pro-angiogenic factors. In addition, we found that miR-149-3p secreted by prostate cancer cells induces DAB2IP downregulation in recipient vascular endothelial cells, stimulating their proliferation and motility, thus potentially remodeling the tumor microenvironment. Finally, we found that inhibition of endogenous miR-149-3p restores DAB2IP activity and efficiently reduces tumor growth and dissemination of malignant cells. These observations suggest that miR-149-3p can promote cancer progression via coordinated inhibition of DAB2IP in tumor cells and in stromal cells.

A BARF1-specific mAb as a new immunotherapeutic tool for the management of EBV-related tumors.

The use of monoclonal antibodies (mAb) for the diagnosis and treatment of malignancies is acquiring an increasing clinical importance, thanks to their specificity, efficacy and relative easiness of use. However, in the context of Epstein-Barr virus (EBV)-related malignancies, only cancers of B-cell origin can benefit from therapeutic mAb targeting specific B-cell lineage antigens. To overcome this limitation, we generated a new mAb specific for BARF1, an EBV-encoded protein with transforming and immune-modulating properties. BARF1 is expressed as a latent protein in nasopharyngeal (NPC) and gastric carcinoma (GC), and also in neoplastic B cells mainly upon lytic cycle induction, thus representing a potential target for all EBV-related malignancies. Considering that BARF1 is largely but not exclusively secreted, the BARF1 mAb was selected on the basis of its ability to bind a domain of the protein retained at the cell surface of tumor cells. In vitro, the newly generated mAb recognized the target molecule in its native conformation, and was highly effective in mediating both ADCC and CDC against BARF1-positive tumor cells. In vivo, biodistribution analysis in mice engrafted with BARF1-positive and -negative tumor cells confirmed its high specificity for the target. More importantly, the mAb disclosed a relevant antitumor potential in preclinical models of NPC and lymphoma, as evaluated in terms of both reduction of tumor masses and long-term survival. Taken together, these data not only confirm BARF1 as a promising target for immunotherapeutic interventions, but also pave the way for a successful translation of this new mAb to the clinical use.

Rational design of polyarginine nanocapsules intended to help peptides overcoming intestinal barriers.

The aim of this work was to rationally design and characterize nanocapsules (NCs) composed of an oily core and a polyarginine (PARG) shell, intended for oral peptide delivery. The cationic polyaminoacid, PARG, and the oily core components were selected based on their penetration enhancing properties. Insulin was adopted as a model peptide to assess the performance of the NCs. After screening numerous formulation variables, including different oils and surfactants, we defined a composition consisting of oleic acid, sodium deoxycholate (SDC) and Span 80. This selected NCs composition, produced by the solvent displacement technique, exhibited the following key features: (i) an average size of 180nm and a low polydispersity (0.1), (ii) a high insulin association efficacy (80-90% AE), (iii) a good colloidal stability upon incubation in simulated intestinal fluids (SIF, FaSSIF-V2, FeSSIF-V2), and (iv) the capacity to control the release of the associated insulin for >4h. Furthermore, using the Caco-2 model cell line, PARG nanocapsules were able to interact with the enterocytes, and reversibly modify the TEER of the monolayer. Both cell adhesion and membrane permeabilization could account for the pronounced transport of the NCs-associated insulin (3.54%). This improved interaction was also visualized by confocal fluorescent microscopy following oral administration of PARG nanocapsulesto mice. Finally, in vivo efficacy studies performed in normoglycemic rats showed a significant decrease in their plasma glucose levels after treatment. In conclusion, here we disclose key formulation elements for making possible the oral administration of peptides.

Design, synthesis and evaluation of biotin decorated inulin-based polymeric micelles as long-circulating nanocarriers for targeted drug delivery.

Here, long-circulating behaviors of Inulin-based nanomicelles are demonstrated for the first time in vivo. We show the synthesis and evaluation of biotin (BIO)-decorated polymeric INVITE micelles constituted of substances of natural origin, Inulin (INU) and Vitamin E (VITE), as long-circulating carriers for receptor-mediated targeted drug delivery. The resulting INVITE or INVITE-BIO micelles, nanometrically sized, did not reveal any cytotoxicity after 24h of incubation with Caco-2 cells. Moreover, in vitro studies on Caco-2 cells monolayers indicated that the transport of INVITE-BIO micelles was faster than surface unmodified INVITE micelles. In vivo optical imaging studies evidenced that, upon intravenous administration, INVITE-BIO micelles were quantitatively present in the body up to 48h. Instead, after oral administration, the micelles were not found in the systemic circulation but eliminated with the normal intestinal content. In conclusion, INVITE-BIO micelles may enhance drug accumulation in tumor-cells over-expressing the receptor for biotin through receptor mediated endocytosis.

Site-Specific Transglutaminase-Mediated Conjugation of Interferon α-2b at Glutamine or Lysine Residues.

Interferon α (IFN α) subtypes are important protein drugs that have been used to treat infectious diseases and cancers. Here, we studied the reactivity of IFN α-2b to microbial transglutaminase (TGase) with the aim of obtaining a site-specific conjugation of this protein drug. Interestingly, TGase allowed the production of two monoderivatized isomers of IFN with high yields. Characterization by mass spectrometry of the two conjugates indicated that they are exclusively modified at the level of Gln101 if the protein is reacted in the presence of an amino-containing ligand (i.e., dansylcadaverine) or at the level of Lys164 if a glutamine-containing molecule is used (i.e., carbobenzoxy-l-glutaminyl-glycine, ZQG). We explained the extraordinary specificity of the TGase-mediated reaction on the basis of the conformational features of IFN. Indeed, among the 10 Lys and 12 Gln residues of the protein, only Gln101 and Lys164 are located in highly flexible protein regions. The TGase-mediated derivatization of IFN was then applied to the production of IFN derivatives conjugated to a 20 kDa polyethylene glycol (PEG), using PEG-NH2 for Gln101 derivatization and PEG modified with ZQG for Lys164 derivatization. The two mono-PEGylated isomers of IFN were obtained in good yields, purified, and characterized in terms of protein conformation, antiviral activity, and pharmacokinetics. Both conjugates maintained a native-like secondary structure, as indicated by far-UV circular dichroism spectra. Importantly, they disclosed good in vitro antiviral activity retention (about only 1.6- to 1.8-fold lower than that of IFN) and half-lives longer (about 5-fold) than that of IFN after intravenous administration to rats. Overall, these results provide evidence that TGase can be used for the development of site-specific derivatives of IFN α-2b possessing interesting antiviral and pharmacokinetic properties.

A site-selective hyaluronan-interferonα2a conjugate for the treatment of ovarian cancer.

While interferon alpha (IFNα) is used in several viral and cancer contexts, its efficacy against ovarian cancer (OC) is far from being incontrovertibly demonstrated and, more importantly, is hindered by heavy systemic side effects. To overcome these issues, here we propose a strategy that allows a targeted delivery of the cytokine, by conjugating IFNα2a with an aldehyde-modified form of hyaluronic acid (HA). The resulting HA-IFNα2a bioconjugate was biochemically and biologically characterized. The conjugation with HA did not substantially modified both the antiviral function and the anti-proliferative activity of the cytokine. Moreover, the induction of STAT1 phosphorylation and of a specific gene expression signature in different targets was retained. In vivo optical imaging biodistribution showed that the i.p.-injected HA-IFNα2a persisted into the peritoneal cavity longer than IFNα2a without being toxic for intraperitoneal organs, thus potentially enhancing the loco-regional therapeutic effect. Indeed, in OC xenograft mouse models bioconjugate significantly improved survival as compared to the free cytokine. Overall, HA-IFNα2a bioconjugate disclosed an improved anticancer efficacy, and can be envisaged as a promising loco-regional treatment for OC.

Drug conjugation to hyaluronan widens therapeutic indications for ovarian cancer.

Management of ovarian cancer still requires improvements in therapeutic options. A drug delivery strategy was tested that allows specific targeting of tumor cells in combination with a controlled release of a cytotoxic molecule. To this aim, the efficacy of a loco-regional intraperitoneal treatment with a bioconjugate (ONCOFID-S) derived by chemical linking of SN-38, the active metabolite of irinotecan (CPT-11), to hyaluronan was assessed in a mouse model of ovarian carcinomatosis. In vitro, the bioconjugate selectively interacted with ovarian cancer cells through the CD44 receptor, disclosed a dose-dependent tumor growth inhibition efficacy comparable to that of free SN-38 drug, and inhibited Topoisomerase I function leading to apoptosis by a mechanism involving caspase-3 and -7 activation and PARP cleavage. In vivo, the intraperitoneal administration of ONCOFID-S in tumor-bearing mice did not induce inflammation, and evidenced an improved therapeutic efficacy compared with CPT-11. In conclusion, SN-38 conjugation to hyaluronan significantly improved the profile of in vivo tolerability and widened the field of application of irinotecan. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of ovarian cancer.

Peritoneal tumor carcinomatosis: pharmacological targeting with hyaluronan-based bioconjugates overcomes therapeutic indications of current drugs.

Peritoneal carcinomatosis still lacks reliable therapeutic options. We aimed at testing a drug delivery strategy allowing a controlled release of cytotoxic molecules and selective targeting of tumor cells. We comparatively assessed the efficacy of a loco-regional intraperitoneal treatment in immunocompromised mice with bioconjugates formed by chemical linking of paclitaxel or SN-38 to hyaluronan, against three models of peritoneal carcinomatosis derived from human colorectal, gastric and esophageal tumor cell xenografts. In vitro, bioconjugates were selectively internalized through mechanisms largely dependent on interaction with the CD44 receptor and caveolin-mediated endocytosis, which led to accumulation of compounds into lysosomes of tumor cells. Moreover, they inhibited tumor growth comparably to free drugs. In vivo, efficacy of bioconjugates or free drugs against luciferase-transduced tumor cells was assessed by bioluminescence optical imaging, and by recording mice survival. The intraperitoneal administration of bioconjugates in tumor-bearing mice exerted overlapping or improved therapeutic efficacy compared with unconjugated drugs. Overall, drug conjugation to hyaluronan significantly improved the profiles of in vivo tolerability and widened the field of application of existing drugs, over their formal approval or current use. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of peritoneal carcinomatosis.

PSMA-specific CAR-engineered T cells eradicate disseminated prostate cancer in preclinical models.

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affinity specific mAb. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter capable of robust and coordinated expression of the CAR molecule, and a bioluminescent reporter gene to allow the tracking of transgenic T cells after in vivo adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC, which in turn were readily stimulated to produce cytokines and to exert a relevant cytotoxic activity by engagement with PSMA+ prostate tumor cells. Upon in vivo transfer in tumor-bearing mice, CAR-transduced T cells were capable to completely eradicate a disseminated neoplasia in the majority of treated animals, thus supporting the translation of such approach in the clinical setting.

Paclitaxel-hyaluronan hydrosoluble bioconjugate: mechanism of action in human bladder cancer cell lines.

OBJECTIVES: A previously described hydrosoluble paclitaxel-hyaluronan bioconjugate appears particularly well suited for treatment of superficial bladder cancer because of its in vitro cytotoxic profile against urothelial carcinoma (UC) cell lines and in vivo biocompatibility. The aim of this work was to assess the mechanism of action of the bioconjugate in UC cells. MATERIALS AND METHODS: Expression of CD44 and RHAMM hyaluronan-binding receptors in RT-4 and RT-112/84 UC cell lines, interaction of fluorochrome-labeled bioconjugate with tumor cells, CD44 modulation upon incubation with the compound or free hyaluronan, and caspase activation were assessed by flow cytometry. Cytotoxicity was studied by the MTT assay. Analysis of bioconjugate intracellular localization and effects on ß-tubulin organization was carried out by confocal microscopy. RESULTS: The paclitaxel-hyaluronan bioconjugate bound to UC tumor cells entered intracellular compartments through a saturable and energy-dependent mechanism that involved CD44, as assessed by blocking with specific antibody. Upon internalization, the bioconjugate accumulated into lysosomes where the esteric bond between paclitaxel and the hyaluronan moiety was cleaved, leading to cytoplasmic diffusion of the free drug, caspase activation, and disruption of the ß-tubulin microtubular mesh with subsequent cell death. CONCLUSIONS: Conjugation of paclitaxel to hyaluronan results in a new chemical entity, characterized by selective targeting to polymer receptors on plasma membrane and cell entry through receptor-mediated endocytosis, followed by lysosomal accumulation. Ultimately, the active molecule is released, fully preserving the cytotoxic potential and profile of clinically used free paclitaxel.