L-lysine Citramide Research Articles

Simple fabrication of multifunctional hyperbranched copolymer based on l-lysine and citric acid for co-delivery of anticancer drugs to breast cancer cells

The aim of this study was to prepare a novel drug delivery system based on hyperbranched polymers having lysine as the repeating units for cancer treatment. The hyperbranched Poly (l-Lysine citramide) (HBPLC) was synthesized from citric acid and l-lysine via the polycondensation method. Then, the post-functionalizing of the desired polymer with both PEG and folic acid was carried out in order to achieve the HBPLC-PEG-FA nanocarrier. The structure and morphology of the resulted system was determined using different techniques. According to SEM studies, the particle sizes of prepared HBPLC and HBPLC-PEG-FA were about 10–15 nm and 80–150 nm, respectively. The loading capacity, in-vitro release behavior, antioxidant, and MTT characteristics of HBPLC-PEG-FA were investigated. The encapsulation efficiency (EE%) values of DOX and MTX for HBPLC-PEG-FA were 96.3% and 96.9%, respectively. The antioxidant activity of the HBPLC-PEG-FA nanocarrier was 53.9%, and the kinetic and in-vitro drug release (RD) studies of nanocarrier exhibited that HBPLC-PEG-FA had controlled pH-responsive behavior (DOX: 92.7% and MTX: 97.02% at pH 5, respectively). In-vitro cytotoxicity results and degradation studies revealed that the synthesized HBPLC-PEG-FA is biocompatible and biodegradable.

Modulation of the electroosmotic mobility using polyelectrolyte multilayer coatings for protein analysis by capillary electrophoresis

Successive multiple ionic-polymer layers (SMIL) coatings have been often used in capillary electrophoresis due to their simplicity to implement and regenerate. However, the performances of the separation are strongly dependent on the nature of the polyelectrolyte partners used to build the SMIL coating. In this work, we investigate new couples of polyelectrolytes that were not tested before: namely, polybrene (PB), quaternized diethylaminoethyl dextran (DEAEDq) and ε-poly(lysine) (ε-PLL), as polycations, in combination with poly(acrylic acid), dextran sulfate, poly(styrenesulfonate), poly(methacrylic acid) and poly(l-lysine citramide), as polyanions. Systematic study of intra- and inter-capillaries repeatabilities/reproducibilities was performed based on the determination of migration time, separation efficiency and electroosmotic mobility. Interestingly, the electroosmotic flow was found to vary with the nature of the polycation on a broad range of electroosmotic mobility decreasing in magnitude in the order of PB>ε-PLL > DEAEDq, whatever the polyanion associated. Application of the coatings to the separation of proteins is illustrated in a 0.5 M acetic acid BGE, including CE-MS separation of ribonuclease B-glycoforms of the same mass (positional or structural isomers).

Experimental and Theoretical Studies of Polyanion–Polycation Complexation in Salted Media in the Context of Nonviral Gene Transfection

Positively and negatively charged molecules, endothelia, and cells play important roles in biological salted aqueous media. This work aimed at studying artificial polyelectrolyte complexes in terms of formation and stability in the context of the increasing interest for the use of polyelectrolyte systems in drug delivery or as polyelectrolyte complexes or polyplexes for gene transfection. The effect of salt concentration on model polyelectrolyte complexes was studied both experimentally and from a theoretical viewpoint. The critical salt concentration at which phase separation appeared when multifunctional polyanions, namely poly(l-lysine citramide) and poly(l-lysine citramide imide) were mixed with poly(l-lysine) showed that salt concentration, degree of polymerization and charge density conditioned the formation and the stability of corresponding polyelectrolyte complexes. Data agreed well with the trends indicated by the theoretical approach and they are discussed in comparison with the case of nonviral transfection using polyplexes.

Roles of hydrophobicity and charge density on the dynamics of polyelectrolyte complex formation and stability under modeled physicochemical blood conditions

To improve the understanding of the physicochemical behavior of polyplexes (DNA-polycation complexes) and to avoid the complexity of blood, the formation and stability of polyelectrolyte complexes of degradable polycations and polyanions, we previously studied under pH, temperature, and ionic strength typical of human blood. In this study, the investigation is extended to polycationic macromolecules added into mixtures of polyanions selected to mimic polyanionic species present in blood. The poly(l-lysine) polycation was added to binary mixtures of degradable polyanions with different charge densities and hydrophobicity. The polyanions were poly(l-lysine citramide imide), poly(l-lysine citramide), and poly(l-lysine citramide) partially esterified with heptyl groups. We found selectivity and fractionation in the molar mass, which depended on the structural characteristics of the polyanions. The affinity of polycationic poly(l-lysine) macromolecules to polyanions increased in the following order: poly(l-lysine citramide imide) < poly(l-lysine citramide) < hydrophobized poly(l-lysine citramide). These data complements the previous information with respect to the possibility of polyplex destabilization and/or the interactions of polycationic macromolecules in excess with the polyanionic species present in blood, depending on the physicochemical characteristics of the polyplex, the excess polycation, and blood elements.

Advanced Functional Polymers for Medicine

Advanced Functional Polymers for Medicine

Intraperitoneal administration of novel doxorubicin loaded polymeric delivery systems against peritoneal carcinomatosis: Experimental study in a murine model of ovarian cancer

Objective Peritoneal spread is an adverse outcome in ovarian cancer. Despite clinical efficiency, intraperitoneal (i.p.) chemotherapy after cytoreductive surgery is associated with high systemic and local toxicity. Two polymer-drug delivery systems (P-HYD1-DOX and P-HYD2-DOX) were developed for i.p. administration by conjugating doxorubicin (DOX) to a poly( l-Lysine citramide) polymer carrier with a hydrazone-based degradable spacer. The aim of this study was to assess the antitumoral efficacy of these two conjugates in a xenograft model of human ovarian carcinomatosis. Methods Peritoneal carcinomatosis was generated in athymic mice by i.p. injection of SKOV3-Luc cells. Free DOX, P-HYD1-DOX and P-HYD2-DOX solutions were administered i.p. at the same dose of 10 mg/kg (DOX eq.). For each treatment, tumor load and therapeutic efficacy were compared to untreated mice and assessed by bioluminescence imaging and survival rates. Toxicity profiles in each group and biodistribution of P-HYD2-DOX after i.p. administration were also determined. Results P-HYD-1-DOX and P-HYD-2-DOX demonstrated significant antitumoral efficacy against peritoneal carcinomatosis. Compared to untreated group, P-HYD1-DOX improved median survival times from 58 to 105 days. For P-HYD2-DOX, median survival was not reached after a follow-up of 120 days. Bioluminescence showed high efficacy of P-HYD-2-DOX compared to free DOX but the difference was not significant. Biodistribution study confirmed that free and active DOX were successively released from P-HYD2-DOX in vivo. P-HYD-DOX conjugates were well tolerated by mice after i.p. injection. Conclusion P-HYD-DOX conjugates demonstrated significant activity against peritoneal carcinomatosis in a xenograft model of ovarian carcinomatosis and their ability to release active DOX in i.p. deposits and tumor. These features are of clinical interest for i.p. administration in the treatment of ovarian peritoneal carcinomatosis after cytoreductive surgery.

Dynamics of polyelectrolyte complex formation and stability as a polyanion is progressively added to a polycation under modeled physicochemical blood conditions

To understand the fate of anionic macromolecular species when injected into blood, poly(acrylic acid) and poly(L-lysine citramide) polyanions, with better charge densities, and the poly(L-lysine) polycation were used as models of negatively charged polymer—drug conjugates and positively charged blood proteins, respectively. To mimic an intravenous injection, the polyanion was added to the poly(L-lysine) stepwise at room temperature. The polyelectrolyte complexes formed as precipitates and the molar mass fractionation was observed from one fraction to the other, especially in the case of largely polydispersed poly(L-lysine). The salt concentration necessary to return each fraction of complexed polyelectrolyte back to solution varied linearly with the logarithm of the molar mass of the polycation component. The physicochemical characteristics data of the polyelectrolytes and the media are compared to previously reported reverse mixing mode when the polycation is introduced into a solution of polyanions.

Dynamics of polyelectrolyte complex formation and stability when a polycation is progressively added to a polyanion under physico-chemical conditions modeling blood

The formation of polyelectrolyte complexes is known to depend on many factors, especially pH, temperature, and ionic strength, as well as acid—base properties and mixing conditions. In an approach aimed at by-passing the complexity of blood, the formation and the stability of complexes between oppositely charged polymers were studied in salted media (0.15N NaCl and 0.13 M, pH 7.4 PBS) at room temperature. Different molar masses of poly(L-lysine) were reacted with polyanions with different chemical structures and charge densities, namely: poly(acrylic acid), poly(L-lysine citramide), poly(L-lysine citramide imide), and poly(malic acid). A stepwise protocol was used to investigate the fractionation phenomena reported previously. After each addition, the precipitate was separated and analyzed. The polyanion macromolecules were fractionated according to their structure; no significant fractionation was observed for the polycation. The NaCl concentration, required to destabilize the complexes in the isolated fractions, was found to depend on the polycation molar mass and to vary linearly with log(polyanion Mw). Based on these data, the possible fate of polycationic species, and of polycation-based polyelectrolytic complex, when injected into blood, are addressed.

Degradability of poly( l-lysine) and poly( dl-aminoserinate) complexed with a polyanion under conditions modelling physico-chemical characteristics of body fluids

Poly( l-lysine), (PLL), and poly( dl-amino serinate), (PSA), are respectively enzymatically and hydrolytically degradable polycations. This work was aimed at investigating their degradability when they are complexed with polyanions, namely poly(acrylic acid) and poly( l-lysine citramide), taken as simple models of DNA in polyplexes. Comparison was made with degradation characteristics of the same polycations in solution in the absence of polyanion on the basis of size exclusion chromatography and capillary zone electrophoresis. Complexed PLL remained enzymatically degradable by trypsin, an endopeptidase, but was no longer degradable by aminopeptidase, an exopeptidase. Trypsin yielded a mixture of trilysin and tetralysin. Complexed PSA remained hydrolytically degradable in aqueous media. The hydrolysis of PSA led to dl-serine. However, traces of anionic species were also detected that were identified as residues of constituting repeating units issued from the N-benzyloxycarbonyl polyaminoserinate precursor (PSAZ).

Peptide−Poly(l-lysine citramide) Conjugates and their In Vitro Anti-HIV Behavior

Poly(L-lysine citramide) is a degradable bioresorbable polyanion whose polyamide chains are composed of citric acid and L-lysine building blocks. Its chemical and physicochemical properties were extensively investigated in the past for its interest as polymeric drug carrier. In this work, 4(S)-amino-3-(S)-hydroxy-5-phenylpentanoyl-isoleucyl-phenylalanine methyl ester, a pseudopeptide active against the HIV protease in vitro, was linked to poly(L-lysine citramide) in attempts to promote solubility and cell penetration. Conjugates were characterized by FTIR, NMR, SEC, DLS, amino-acid analyses, and toxicity in mice. They degraded slowly at pH 7.4 and more rapidly at pH 4.5, two pH values selected to mimic extra-cellular fluids and intralysosome medium, respectively. According to capillary zone electrophoresis, degradation did not release the peptide. The phenylalanyl-isoleucyl-phenylalanine methyl ester peptide, inactive against the protease in vitro, was used as negative control. The anti-HIV activities of the carrier, of the conjugates and of model molecules, including a fluorescence-labeled pseudopeptide conjugate, were evaluated comparatively in vitro using two cell lines, namely, CEM-SS and MT-4 cells, infected with HIV-1 LAI and IIIB isolates, respectively. Unexpectedly, all the conjugates showed in vitro antiviral activity independent of peptide release and of inhibition of the HIV protease. According to FACS analysis, the antiviral activity was related to the presence of peptide moieties along the polymer chains and depended on the order by which cells, viruses, and conjugates were presented to each other. Although it was not possible to determine whether the antiviral activity resulted from interactions between conjugates and cells or conjugates and virus or both, the conjugates appeared able to inhibit the binding of the virus to cells in vitro when introduced before cell infection. None of the conjugates exhibited acute toxicity in mice.

Study of Interpolymer Complexes of Oppositely Charged Macromolecules with Different Affinity to Solvent

We consider the new type of water-soluble stoichiometric interpolymer polyelectrolyte complexes (IPEC) consisting of macromolecules having different affinities to solvent and different densities of ionic groups along the chain. It was proposed that complexes of such macromolecules have a core−shell structure. The inner part of complex with a radius r contains monomer units of both macromolecules, while exterior part with a radius R consists exclusively of monomer units of hydrophilic macromolecules. With such complex organization, the external charged hydrophilic shell effectively protects the complex from precipitation and fusion with other complex assemblies. These theoretical conclusions allow to explain the experimental data on the stability of IPECs containing poly(l-lysine), poly(l-lysine citramide), poly(l-lysine citramide imide), and poly(acrylic acid), and can be of interest in the field of DNA−polycation complexes aimed at transfecting genes to cell nuclei.

Biodistribution of doxorubicin-alkylated poly( l-lysine citramide imide) conjugates in an experimental model of peritoneal carcinomatosis after intraperitoneal administration

Peritoneal spread is a common manifestation in ovarian and gastrointestinal cancer. Intraperitoneal (i.p.) chemotherapy after cytoreductive surgery is associated with high systemic or local toxicity. A macromolecular drug delivery system was evaluated with the aim of improving the therapeutic index of i.p. chemotherapy. Peritoneal carcinomatosis was generated in BDIX rats ( n = 55) by i.p. injection of 2 million DHDK12 cells. Fourteen days later, doxorubicin (DOX) and two DOX-alkylated poly( l-lysine citramide imide) conjugates bearing 9.5% and 20.5% (w/w) chemically bound drug, respectively, were given i.p. to rats at a single 2 mg DOX/kg dose. Free and polymer-bound DOX were assessed in plasma, peritoneal fluid, abdominal tissues and heart, 15 min, 2, 6, 24, 48 and 168 h after injection. According to pharmacokinetic profiles, the peritoneal fluid areas under the concentration versus time curves (AUCs) were 2 and 2.6 times greater for the conjugates (P-DOX20 and P-DOX10, respectively) than for the free drug, respectively. Conjugates crossed the peritoneal barrier slower than the free drug. For the tumor, AUCs were, respectively, 3 and 7 times higher for the conjugates than for free DOX. The elimination half-lives of the conjugates were higher than that calculated for the free drug. Only very small concentrations were detected in peripheral organs and in the heart. In contrast, significant retention and accumulation of the conjugates were found in abdominal organs, particularly in the tumor. There was no sign of macroscopic chemical peritonitis after injection of the polymer–DOX conjugates. In conclusion, the conjugates have higher elimination half-lives than free DOX and were preferentially retained in abdominal organs and in the peritoneal carcinomatosis. This feature is of clinical interest to target tumor deposits.

Release of Polyanions from Polyelectrolyte Complexes by Selective Degradation of the Polycation

One of the major problems associated with analyzing polyelectrolyte complexes is the separation of strongly bound oppositely charged polymeric components. As part of a work aimed at better understanding the factors that affect polyelectrolyte complex formation and stability, an investigation of the possibility to release and analyze the polyanion, after hydrolytic or enzymatic degradation of the partner polycation, was made. Mixtures of poly(acrylic acid) or poly(L-lysine citramide) polyanions with poly(L-lysine) or poly(amino serinate) polycations were investigated. For each polycation-polyanion couple, four complex fractions were obtained by adding the polycation to the polyanion according to a titration protocol. The selective degradation of the polycation within the different complex fractions was investigated after the complex was disrupted with a NaCl solution. The molecular weights of the recovered polyanionic macromolecules were assessed by both static light scattering and size exclusion chromatography. The data supported previous findings that complexation was selective according to the molecular weight of the polyanion for a given polycation. The lower the degree of neutralization of the polyanion negative charges by the polycation positive charges, the greater the molecular weight of the complexed polyanionic macromolecules.

Polyelectrolyte complex formation and stability when mixing polyanions and polycations in salted media: A model study related to the case of body fluids

Controlled drug delivery and gene transfection involve contact of artificial polyelectrolytic systems that can interact dramatically with biopolymers and cells when they are introduced in blood. Given the complexity of body aqueous media in terms of physical chemistry, a model approach was selected in attempt to understand the behavior of artificial polyelectrolytes introduced in body fluids. Selection in terms of molecular weight was highlighted in a previous paper. In the present study the formation and the stability of fractions obtained when a polycation is added to a polyanion according to a titrating process mimicking injection into blood was considered for different polycation/polyanion couples. Poly(amino serinate) and poly( l-lysine) were used as polybases, and poly(acrylic acid), poly( l-lysine citramide) and poly( l-lysine citramide imide) as polyacids. Four fractions corresponding to different positive/negative charge ratios were formed for each couple. At low polyion concentration (13 mg/L) and given salt concentration, the stability of the complex fractions depended on molecular weight and charge density of the polyions. The NaCl concentration required to destabilize the different interpolyelectrolyte complexes was found to decrease from the first fraction to the fourth one. Upon decreasing the salt concentration, macroscopic flocculation occurred in the case of PLL/PAA complex fractions only. For the other couples, dynamic light scattering showed that several hundreds nanometer sized particles were formed that were stable in a broad range of NaCl concentration, including the physiological 0.15 ionic strength. At higher polyion concentrations, stable solid precipitate was formed regardless of the system. The absence of flocculation in the case of highly diluted poly( l-lysine citramide) and poly( l-lysine citramide imide) polyanions in salted media is assigned to the presence of non-ionic hydroxyl and amide polar groups along the complexed chains. Data show that introducing non-ionic functions along the polyelectrolyte chains is a good means to keep interpolyelectrolyte complexes dispersed in salted media, a conclusion of interest in the field of condensation of genes by polycations.

Norfloxacin-Poly(l-Lysine Citramide Imide) Conjugates and Structure-dependence of the Drug Release

Norfloxacin (Nflx), an antibiotic which is active against some intracellular bacteria, was coupled to a polymeric carrier, namely poly(l-lysine citramide) via a lysine or an ethylcarbamate spacer to obtain a macromolecular prodrug. The carrier, which derived from the two metabolites citric acid and l-lysine, is known to be biocompatible and slowly degradable under slight acidic conditions. Conjugates were characterised by UV, 1H and 13C NMR and SEC. The presence of Norfloxacin and the lysine type spacer caused chain aggregation, due to a probable physical cure. The release of Norfloxacin from these prodrugs and from a prodrug where Norfloxacin is bound to the carrier backbone without spacer arm was investigated comparatively in vitro. Conjugation via a carbamate-type linkage appeared as a method to achieve the release of Norfloxacin from a PLCA-type conjugate at neutral.

Affinity Chromatography as a Tool to Analyze Polyanion–Polycation Complexes: the Case of Poly(Llysine Citramide)–Poly(L-lysine) Systems

Artificial polymeric carriers for bioactive compounds, such as drugs and genes, are increasingly being reported in the literature. Most are polyelectrolytes aimed at being injected into body fluids that are composed of charged macromolecules (proteins, glycoproteins, poly(glycosamino glycane)s, polynucleotides, etc.). Many interactions can occur and lead to dramatic phenomena, such as polyelectrolyte complexation or substitution, cell aggregation and hemolysis. For the purpose of modeling and better understanding these interactions and their effects, a library of polyanions of the poly(L-lysine citramide)-type was interacted with the polycation poly(L-lysine) in aqueous media. Different fractions of complexes were generated by successive additions of aliquots of the polycation solution to the polyanion library solution by a titrating process. The two components, the polycation and the complex population of polyanions, were separated from the collected complex fractions by affinity chromatography and then analyzed by aqueous SEC. Molecular weight selectivity was shown, the high molecular weight polyanion macromolecules precipitated first, while complexes involving smaller molecules were found to remain in solution. The data show the potential of affinity chromatography for studying interpolyelectrolyte complexes and emphasizes the critical role that polydispersity can play when therapeutic charged macromolecules are injected into blood.

Molecular Microencapsulation: Paclitaxel Formations in Aqueous Medium Using Hydrophobized Poly(L-Lysine Citramide Imide)

Hydrophobized polyelectrolytes are able to aggregate in aqueous media and to form lipophilic microdomains which are much more stable than micelles made of amphiphilic diblock copolymers. Solubilization of paclitaxel in water by molecular encapsulation was attempted in the hydrophobic pockets present in the aggregates of hydrophobized poly(l-lysine citramide imide) polymers, PLCAixRy, where x = percentage in imide groups and y = percentage in alkyl substituents per lysine citramide repeat unit. A comparison was made of the physical entrapment and the solubilization of paclitaxel in PLCAi27C1234, PLCAC1275, PLCAi13C730, PLCAC12100 aggregates and in the usual allergic Cremophor® present in the Taxol® commercial formulation. PLCAixRy polymers solubilized paclitaxel in the selected aqueous medium proportionally to the polymer concentration. For comparable amounts in weight, the polymers were two to six times less efficient than Cremophor® in terms of equivalent mass of solubilizing species. A comparison between PLCAi13C730 and PLCAixC12y polymers suggested that dodecyl hydrophobizing groups were more efficient than heptyl ones in promoting the physical entrapment of paclitaxel. The antitumoral activity of the polymer-solubilized paclitaxel was tested in vitro against an ovarian carcinoma cell line (A2780). The C12 systems showed cytotoxicity, the IC50 values were statistically comparable in the 6.5-10.5 μg/L range. However, the amounts of drug incorporated in the polymer aggregates at the tested concentrations were still too low for clinical use. Improvements are expected from PLCA polymers with longer alkyl chains, greater hydrophobicity and higher concentrations.

Alkylated poly(l-lysine citramide) as models to investigate the ability of amphiphilic macromolecular drug carriers to physically entrap lipophilic compounds in aqueous media

Poly(l-lysine citramide) was synthesized to serve as a polymeric bioresorbable drug carrier. It was previously shown that low molecular weight poly(l-lysine citramide) hydrophobized with heptyl and lauryl side chains (PLCA-C7p with p=43 and 60%; and PLCA-C12p, with p=68, 75 and 100%) formed aggregates in aqueous media. The size of these aggregates was found to depend on the balance between repulsive electrostatic charges and attractive hydrophobic interactions, on the degree of ionization, and on the ionic strength. In this paper, the formation of these aggregates was further investigated by fluorescence probing, using two polarity sensitive molecules, pyrene and Nile Red, which were physically entrapped within the lipophilic core of the aggregates. In contrast to other micellar structures formed by surfactants and amphiphilic block copolymers, aggregates were observed even at very low polymer concentrations. The capacity of the hydrophobic domains to accommodate lipophilic molecules via physical entrapment was demonstrated with progesterone.

Synthesis, Characterisation and In Vivo Behaviour of a Norfloxacin-Poly(L-Lysine Citramide Imide) Conjugate Bearing Mannosyl Residues

With the aim of promoting the targeting of macrophage mannose receptors and the internalisation of the norfloxacin antibiotic, which is active against some intracellular bacteria, a macromolecular prodrug was synthesised where the antibiotic and mannosyl moieties were coupled to a polymeric carrier, namely poly(L-lysine citramide imide). This carrier, which derived from two metabolites, citric acid and L-lysine, is known to be biocompatible and slowly degradable under slight acidic conditions. Norfloxacin was coupled onto the acid groups present along the polymer chains, and conjugates were characterised by UV, TLC and SEC. The mannosyl groups selected to promote the targeting of the mannose-specific lectin present on the outer membrane of macrophages were incorporated through a biodegradable glycolic spacer arm. Two different strategies were considered to synthe-sise the full conjugates, namely coupling norfloxacin onto mannosylated conjugates, and coupling mannose onto PLCAI/Nflx conjugates. The second pathway led to better results regarding mannosylation. The presence of norfloxacin and mannose caused chain aggregation, especially for conjugates with a high content of mannosyl residues. The targeting ability of the prodrug was investigated using a method based on the competition between the mannosylated macromolecules and glucose oxidase, a mannosyl-bearing non-human protein. This method showed that prodrug macromolecules competed effectively with glucose oxidase and thus should be able to bring the drug up to the mannosyl receptor-bearing membranes of macrophages infected by intracellular bacteria.

Metabolite-derived artificial polymers designed for drug targeting, cell penetration and bioresorption

The use of poly(β-malic acid) and poly( l-lysine citramide) carriers to transport doxorubicin (Dox) within K562 myeloblastic cells was studied by taking advantage of laser microspectrofluorometry (L-MSF). This technique provided a means to monitor and to quantify the penetration of Dox molecules in the cytoplasm and in the nucleus of Dox-sensitive and Dox-resistant cells. Comparison was made between polymer-drug conjugates more or less hydrophobised by C2, C7 and C12 aliphatic substituents and by the Dox attached to the polymer backbone as pendent chains. Furthermore, a method was proposed to introduce a cleavable non-peptidic spacer of the lactyllactyl-type between the poly( l-lysine citramide) backbone and the drug. It is shown that: Dox was released from the conjugates by a non-enzymatic route in the absence of cells, the Dox-uptake by cells was slower for the conjugates than for the free drug, and the hydrophobisation promoted the penetration of the released drug within the nucleus, even in the case of Dox-resistant cells. However, no reversion of the resistance was observed.