Antitumor efficacy following the intracellular and interstitial release of liposomal doxorubicin

pH-triggered lipid-membranes designed from biophysical principles are evaluated in the form of targeted liposomal doxorubicin with the aim to ultimately better control the growth of vascularized tumors. We compare the antitumor efficacy of anti-HER2/neu pH-triggered lipid vesicles encapsulating doxorubicin to the anti-HER2/neu form of an FDA approved liposomal doxorubicin of DSPC/cholesterol-based vesicles. The HER2/neu receptor is chosen due to its abundance in human breast cancers and its connection to low prognosis. On a subcutaneous murine BT474 xenograft model, superior control of tumor growth is demonstrated by targeted pH-triggered vesicles relative to targeted DSPC/cholesterol-based vesicles (35% vs. 19% decrease in tumor volume after 32 days upon initiation of treatment). Superior tumor control is also confirmed on SKBR3 subcutaneous xenografts of lower HER2/neu expression. The non-targeted form of pH-triggered vesicles encapsulating doxorubicin results also in better tumor control relative to the non-targeted DSPC/cholesterol-based vesicles (34% vs. 41% increase in tumor volume). Studies in BT474 multicellular spheroids suggest that the observed efficacy could be attributed to release of doxorubicin directly into the acidic tumor interstitium from pH-triggered vesicles extravasated into the tumor but not internalized by cancer cells. pH-triggered liposome carriers engineered from gel-phase bilayers that reversibly phase-separate with lowering pH, form transiently defective interfacial boundaries resulting in fast release of encapsulated doxorubicin. Our studies show that pH-triggered liposomes release encapsulated doxorubicin intracellularly and intratumorally, and may improve tumor control at the same or even lower administered doses relative to FDA approved liposomal chemotherapy.     

Confrontation of immunohistochemistry and fluorescent in situ hybridization for the assessment of HER-2/<Emphasis Type="Italic">neu</Emphasis> (c-<Emphasis Type="Italic">erb</Emphasis>b-2) status in urothelial carcinoma

Specific treatments targeted toward oncogenes expressed in cancer cells are currently under development. Patients with urothelial carcinomas showing HER-2/neu (human epidermal growth factor receptor 2) overexpression are candidates for such a specific treatment (trastuzumab). However, to be effective, this therapeutic approach requires an extremely reliable evaluation of HER-2/neu status in tumors. In order to assess the status of expression of this gene and to optimize its assessment, we analyzed a series of 64 primary urothelial carcinomas using immunohistochemistry (IHC) with the CB11 monoclonal antibody coupled with fluorescent in situ hybridization (FISH) in 21 cases. Strong HER-2/neu overexpression was detected using IHC in 15 of the 64 (23%) cases analyzed, and this rate rose to 33% for patients with metastases. HER-2/neu overexpression, as revealed using IHC, is strongly associated (95%) with gene amplification assessed using FISH. Patients with urothelial carcinomas overexpressing HER-2/neu using IHC are potential candidates for targeted chemotherapy.     

Specific targeting to B cells by lipid-based nanoparticles conjugated with a novel CD22-ScFv

The CD22 antigen is a viable target for therapeutic intervention for B-cell lymphomas. Several therapeutic anti-CD22 antibodies as well as an anti-CD22-based immunotoxin (HA22) are currently under investigation in clinical settings. Coupling of anti-CD22 reagents with a nano-drug delivery vehicle is projected to significantly improve treatment efficacies. Therefore, we generated a mutant of the targeting segment of HA22 (a CD22 scFv) to increase its soluble expression (mut-HA22), and conjugated it to the surface of sonicated liposomes to generate immunoliposomes (mut-HA22-liposomes). We examined liposome binding and uptake by CD22⁺ B-lymphocytes (BJAB) by using calcein and/or rhodamine PE-labeled liposomes. We also tested the effect of targeting on cellular toxicity with doxorubicin-loaded liposomes. We report that: (i) Binding of mut-HA22-liposomes to BJAB cells was significantly greater than liposomes not conjugated with mut-HA22 (control liposomes), and mut-HA22-liposomes bind to and are taken in by BJAB cells in a dose and temperature-dependent manner, respectively; (ii) This binding occurred via the interaction with the cellular CD22 as pre-incubation of the cells with mut-HA22 blocked subsequent liposome binding; (iii) Intracellular localization of mut-HA22-liposomes at 37 °C but not at 4 °C indicated that our targeted liposomes were taken up through an energy dependent process via receptor-mediated endocytosis; and (iv) Mut-HA22-liposomes loaded with doxorubicin exhibited at least 2-3 fold more accumulation of doxorubicin in BJAB cells as compared to control liposomes. Moreover, these liposomes showed at least a 2-4 fold enhanced killing of BJAB or Raji cells (CD22⁺), but not SUP-T1 cells (CD22⁻). Taken together these data suggest that these 2nd-generation liposomes may serve as promising carriers for targeted drug delivery to treat patients suffering from B-cell lymphoma.     

Magnetization transfer in liposome and proteoliposome samples that mimic the protein and lipid composition of myelin

Although magnetization transfer (MT) has been widely used in brain MRI, for example in brain inflammation and multiple sclerosis, the detailed molecular origin of MT effects and the role that proteins play in MT remain unclear. In this work, a proteoliposome model system was used to mimic the myelin environment and to examine the roles of protein, cholesterol, brain cerebrosides, and sphingomyelin embedded in the liposome matrix. Exchange parameters were determined using a double‐quantum filter experiment. The goal was to determine the relative contributions to exchange and MT of cerebrosides, sphingomyelin, cholesterol, and proteins in 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine bilayers. The main finding was that cerebrosides produced the strongest exchange effects, and that these were even more pronounced than those found for proteins. Sphingomyelin (which also has exchangeable groups at the head of the fatty acid chains, albeit closer to the lipid acyl chains) and cholesterol showed only minimal transfer. Overall, the extracted exchange rates appeared much smaller than commonly assumed for ‐OH and ‐NH groups.     

The neu-protein and breast cancer

The neu-protein is overexpressed in about 20% of invasive duct cell carcinomas of the breast. The only reliable sign for neu-overexpression by immunohistochemistry is membrane staining. Its overexpression is correlated with decreased overall survival and disease free survival due to increased metastatic activity of neu-overexpressing tumour cells. This increased metastatic potential is a consequence of the motility enhancing activity of the neu-protein, which is exclusively expressed on pseudopodia, and to a lesser extent of its growth stimulating effect. From a clinical point of view, the assessment of neu-overexpression in breast cancer might become a useful tool in the future treatment of patients by chemotherapy, since patients whose tumour shows neu-overexpression benefit from higher doses of chemotherapy. The molecule plays a key role in the pathogenesis of Paget's disease of the breast. A chemotactic factor which is secreted by epidermal keratinocytes attracts the Paget cells to spread into the epidermis and acts via the neu-protein. In ductal carcinoma in situ, the combination of neu-overexpression and large cell type is highly correlated with extent of disease and therefore neu-overexpression might be a predictive marker for recurrence of disease after tumour resection.     

Mixed-chain phospholipids and interdigitated bilayer systems

Summary The lipid bilayer is a fundamental structural component for all biological membranes. The function of lipid bilayers may be considered not only to serve as a permeability barrier preventing the free flows of ions and polar molecules between the cell interior and its external environment, but also to modulate the activity of bilayer-spanning proteins or glycoproteins in biological membranes. Based on the location of the terminal methyl groups of the two hydrocarbon chains in phospholipids or glycosphingolipids, two broad categories of lipid bilayers have been recognized: (1) the non-interdigitated and (2) the interdigitated bilayers. Depending on the chain-length difference between the two hydrocarbon chains, three different types of packing models for interdigitated bilayer systems have been identified. Among the three types, the mixed interdigitated bilayer is perhaps unique, since the hydrocarbon chains in the bilayer core are observed to be interdigitated in both the gel and the liquid-crystalline states. In this communication, various experimental data supporting the chain packing characteristics of the mixed interdigitated bilayer for a large number of mixed-chain phospholipid species are considered. In addition, two types of phase diagrams for binary phospholipid mixtures obtained with mixed-chain phospholipids are also presented. These studies may be of great importance in understanding the functional control of bilayer-spanning proteins in biological membranes, and for providing basic information explaining the dynamic regulation of membrane activities in general.Abbreviations C(X):C(Y)PC saturated L--phosphatidyl-choline having x carbons in thesn-1 acyl chain and Y carbons in thesn-2 acyl chain - DSC differential scanning calorimetry; - T_m the main phase transition temperature Heinrich Wieland-Prize Lecture delivered on October 27, 1989     

Interaction of the anticancer agent Taxol (paclitaxel) with phospholipid bilayers.

Taxol (paclitaxel), a promising agent for use in ovarian and breast cancer, was incorporated into lipid vesicles (liposomes) composed of different saturated and unsaturated phosphatidylcholines, as well as saturated phosphatidylcholines mixed with the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) at different molar ratios, to yield information about Taxol-liposome interactions. For the physicochemical characterization of the thermodynamic, structural, and dynamic properties of these mixtures, differential scanning calorimetry (DSC), steady-state fluorescence depolarization, and Fourier transform IR spectroscopy was used. Time-dependent DSC measurements on 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/Taxol mixtures of different concentrations were performed to yield information on the long-term stability of Taxol-liposome complexes. Partitioning of Taxol into saturated lipid bilayers results in changes of membrane physical properties, such as phase transition temperatures and lipid order parameter, that are different from those observed for unsaturated and charged phospholipid bilayers. Taxol incorporated into saturated phospholipids changes their thermotropic phase behavior: it reduces the lipid order parameter (i.e., has a "fluidizing" effect) in the gel phase of the lipid bilayers. On the contrary, partitioning of Taxol into unsaturated fluid phospholipid bilayers has a slight "rigidization" effect. The saturated lipid bilayer systems DPPC and 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/DMPS have been identified with the highest incorporation efficiency for Taxol and are thus candidates for drug vehicles that can improve the therapeutic efficacy of Taxol.     

Overcoming drug-resistant lung cancer by paclitaxel loaded dual-functional liposomes with mitochondria targeting and pH-response

Mitochondrion-orientated transportation of smart liposomes has been developed as a promising strategy to deliver anticancer drugs directly to tumor sites, and these have a tremendous potential for killing cancer cells, especially those with multidrug resistance (MDR). Herein we report a novel dual-functional liposome system possessing both extracellular pH response and mitochondrial targeting properties to enhance drug accumulation in mitochondria and trigger apoptosis of drug-resistant cancer cells. Briefly, peptide _D[KLAKLAK]₂ (KLA) was modified with 2, 3-dimethylmaleic anhydride (DMA) and combined with 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) to yield a DSPE-KLA-DMA (DKD) lipid. This dual-functional DKD was then mixed with other commercially available lipids to fabricate liposomes. In vitro anticancer efficacy of this liposome system was evaluated in human lung cancer A549 cells and drug-resistant lung cancer A549/Taxol cells. At tumor extracellular pH (∼6.8), liposomes could reverse their surface charge (negative to positive), facilitating liposome internalization. After cellular uptake, KLA peptide directed delivery-enabled selective accumulation of these liposomes into mitochondria and favored release of their cargo paclitaxel (PTX) into desired sites. Specifically, enhanced apoptosis of MDR cancer cells through mitochondrial signaling pathways was evidenced by release of cytochrome c and increased activity of caspase-9 and -3. These dual-functional liposomes had the greatest efficacy for treating A549 cells and A549/Taxol cells in vitro, and in treating drug-resistant lung cancer A549/Taxol cells xenografted onto nude mice (tumor growth inhibition 86.7%). In conclusion, dual-functional liposomes provide a novel and versatile approach for overcoming MDR in cancer treatment.     

Exchange of a spin-labeled analog of PAF-acether between micelles,serum albumin and model membranes

The paper describes the synthesis of an analog of PAF-acether which contains a short spin-labeled acyl chain at position 2. This product exchanges freely between serum albumin, model membranes and micelles (CMC <0.3 M). It mixes as easily with rigid or fluid membrane regions. The probe is localized near the interface between lipid or protein and water.     

Uptake of [3H]-gangliosides by an intestinal protozoan, Giardia lamblia

Because of their limited lipid synthesis ability it has been postulated that Giardia lamblia trophozoites depend on lipid remodeling reactions, to generate parasite-specific phospho and glycolipids. We have shown earlier that exogenous bile acids and lipid molecules are taken up by Giardia through active transport and by other mechanisms. Another report suggests that lipoprotein-like receptors may be present in this parasite that are involved in lipid endocytosis. In the present investigation, we asked whether and by what mechanism exogenous gangliosides can be taken up by Giardia. We found that ganglioside uptake was dependent, on time and concentration, but was not temperature- or carrier-dependent. Gangliosides encapsulated into liposomes were taken up much more effectively than non-encapsulated gangliosides. These results are consistent with a passive uptake process, facilitated by membrane fusion with lipid carriers, but not dependent on a catalytic carrier mechanism. This observation may have some therapeutic implications.     

Uptake of cationized ferritin by colonic epithelium

Guinea-pig, rat and mouse were used in this electron microscopic study to demonstrate endocytosis in absorptive and goblet cells of the surface epithelium of the colon. Cationised ferritin was used as the electron dense tracer which attached in vivo to negatively charged membrane components. Both coated and uncoated vesicles entered the cells. The major pathway for the vesicles was to the secondary lysosomes and occurred within 10–30 min. This demonstrates a new pathway of absorption in the adult rodent colon with potential for the uptake of macromolecules. It may provide new clues to the immunology, physiology and pathology of the tissue.     

Prophylaxis and treatment of experimental lung metastases in mice after treatment with liposome-encapsulated 6-O-stearoyl-N-acetylmuramyl-L-α-aminobutyryl-D-isoglutamine

A new lipophilic muramyl dipeptide analog, 6-O-stearoyl-N-acetylmuramyl-L--aminobutyryl-D-isoglutamine, when incorporated in liposomes, was effective in both the prevention and eradication of experimental pulmonary metastases in mice. Multilamellar vesicles composed of synthetic phospholipids (phosphatidylglycerol and phosphatidylcholine) containing saturated myristoyl or unsaturated dioleoyl acyl chains were found to potentiate the antimetastatic activity of this glycopeptide. Prophylactic and therapeutic efficacy was observed against the three murine tumors tested: FSa, an immunogenic fibrosarcoma; NFSa, a nonimmunogenic fibrosarcoma; and B16 melanoma. Neither the administration of empty liposomes or free glycopeptide, nor their coadministration, had a significant antimetastatic effect. This approach is promising for the therapy of cancer metastases in humans, particularly in the prevention of metastatic seeding and in the treatment of micrometastases.This is contribution No. 180 from the Institute of Bio-Organic Chemistry, Syntex Research.     

Storage stability of optimal liposome–polyethylenimine complexes (lipopolyplexes) for DNA or siRNA delivery

The delivery of nucleic acids such as DNA or siRNA still represents a major hurdle, especially with regard to possible therapeutic applications in vivo. Much attention has been focused on the development of non-viral gene delivery vectors, including liposomes or cationic polymers. Among them, polyethylenimines (PEIs) have been widely explored for the delivery of nucleic acids and show promising results. The combination of cationic polymers and liposomes (lipopolyplexes) for gene delivery may further improve their efficacy and biocompatibility, by combining the favourable properties of lipid systems (high stability, efficient cellular uptake, low cytotoxicity) and PEIs (nucleic acid condensation, facilitated endosomal release). In this study, we systematically analyse various conditions for the preparation of liposome–polyethylenimine-based lipopolyplexes with regard to biological activity (DNA transfection efficacy, siRNA knockdown efficacy) and physicochemical properties (size, zeta potential, stability). This includes the exploration of lipopolyplex compositions containing different liposomes and different relevant branched or linear low-molecular-weight PEIs. We establish optimal parameters for lipopolyplex generation, based on various PEIs, N/P ratios, lipids, lipid/PEI ratios and preparation conditions. Importantly, we also demonstrate that certain lipopolyplexes retain their biological activity and physicochemical integrity upon prolonged storage, even at 37 °C and/or in the presence of serum, thus providing formulations with considerably higher stability as compared to polyplexes. In conclusion, we establish optimal liposome–polyethylenimine lipopolyplexes that allow storage under ambient conditions. This is the basis and an essential prerequisite for novel, promising and easy-to-handle formulations for possible therapeutic applications.     

Absence of amplification of HER-2/neu (c-erbB-2) gene in Ewing's sarcoma: a real-time polymerase chain reaction method

Amplification and overexpression of the HER-2/neu (c-erbB-2) oncogene have been observed in many cancers and are associated with a poor prognosis particularly in breast cancer. The human epidermal growth factor (HER)-2 receptor has recently been implicated in Ewing's sarcoma tumor cell line growth and chemosensitivity. The present study evaluates the amplification of HER-2/neu gene in paraffin sections from 42 cases of Ewing's sarcoma by a real-time quantitative polymerase reaction method using LightCycler system (Roche diagnostics, GmbH Mannheim, Germany). The relative copy number of HER-2/neu versus β-globin was calculated at the crossing point. The mean calculated copy number in these cases of Ewing's sarcoma and normal controls was 26.43 and 26.93, respectively. The p value was 0.215 (p<0.05). Our results demonstrated an absence of HER-2/neu oncogene amplification in Ewing's sarcomas, and we suggest that HER-2/neu is not a biologically or therapeutically important pathway in Ewing's sarcoma.     

Increase in sarcolysin antitumor activity by transforming it into a lipid derivative and incorporation in the membrane of liposomes containing a carbohydrate vector

Lipid derivative of sarcolysin obtained by condensation of sarcolysin with dioleoylglycerol is well retained in the liposome membrane due to high hydrophobic activity exhibits a higher cytotoxic activity than sarcolysinin vitro (CaOv cells) and a much higher antitumor activityin vivo (P388 leukemia). Further increase in the efficacy of the lipid derivativein vivo is attained by incorporating a carbohydrate vector in the liposomal membrane. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 123, No. 4, pp. 439–441, April, 1997     

Dynamics of antibody- and lectin-mediated endocytosis of hapten-containing liposomes by murine macrophages

The uptake by murine macrophages of liposomes, exhibiting one of a variety of haptenic groups on their surfaces, was greatly enhanced by the addition of an intact antibody or a lectin specific for the incorporated hapten. The uptake of untreated liposomes was slow and linear over long periods, whereas upon addition of the antibody or lectin, over 30-fold increase in the maximal rate of uptake was observed. The process reached a plateau after 90–120 min. The interaction of the antibody- or lectintreated liposome with the macrophages apparently resulted in an active endocytosis of the vesicles. As observed by fluorescence microscopy, the distribution of a water-soluble fluorescent, intraliposomal marker had a granular intracellular pattern in treated cells. The uptake was sensitive to azide and the liposome constituents could not be detected at the cell surface. The size of the liposomes as well as the state of stimulation of the macrophages (thioglycollate stimulated vs. normal) did not seem to have a major effect on the phagocytic process. The time required to reach the plateau in uptake was independent of liposome composition or antibody concentration and is, apparently, an intrinsic property of the cells. The implication of this phenomenon on the dynamics of the relevant macrophage receptors is discussed.     

Involvement of HER-2/<Emphasis Type="Italic">neu</Emphasis> and metastasis-related proteins in the development of ileal neuroendocrine tumors

HER-2/neu overexpression and/or gene amplification occurs in several human malignancies, frequently correlates with tumor aggressiveness, and provides the basis for treatment with trastuzumab. Among neuroendocrine neoplasms (NEN) of the gastroenteropancreatic (GEP) tract, ileal neuroendocrine tumors show peculiar features of malignancy with frequent metastases at the diagnosis. We investigated the overexpression and/or amplification of HER-2/neu and the involvement of the metastasis-related proteins c-Met, MTA-1, and VEGF in 24 primary ileal NEN by immunohistochemistry and fluorescence in situ hybridization (FISH). Data were compared with those of 43 GEP endocrine tumors of other sites. All primary ileal NEN showed an intense membranous and cytoplasmic immunostaining for HER-2/neu. According to the breast cancer scoring system, 17% of ileal carcinoids showed a score of 3+ and 71% with a score of 2+ with a significant difference respect the non-ileal GEP endocrine tumors (p < 0.0000). FISH analysis revealed chromosome 17 polysomy in 33% of 2+/3+ ileal tumors but not HER-2/neu gene amplification. The c-Met and MTA-1 but not VEGF were overexpressed in almost all ileal NEN, whereas VEGF presented more frequently a normal staining. The comparisons with the other GEP NEN demonstrated significant differences for all the three proteins (p < 0.0000, p < 0.0002, and p < 0.001, respectively). These findings suggest that in ileal NEN, HER-2/neu overexpression plays a role in the carcinogenetic process and by triggering the altered expression of c-Met and MTA-1, may activate the molecular pathway(s) promoting tumor progression and metastasis development. Ileal HER-2/neu overexpressing neuroendrocrine tumors may constitute potential candidates for target therapy with specific humanized monoclonal antibodies.     

The antimicrobial activity of liposomal lauric acids against Propionibacterium acnes

This study evaluated the antimicrobial activity of lauric acid (LA) and its liposomal derivatives against Propionibacterium acnes (P. acnes), the bacterium that promotes inflammatory acne. First, the antimicrobial study of three free fatty acids (lauric acid, palmitic acid and oleic acid) demonstrated that LA gives the strongest bactericidal activity against P. acnes. However, a setback of using LA as a potential treatment for inflammatory acne is its poor water solubility. Then the LA was incorporated into a liposome formulation to aid its delivery to P. acnes. It was demonstrated that the antimicrobial activity of LA was not only well maintained in its liposomal derivatives but also enhanced at low LA concentration. In addition, the antimicrobial activity of LA-loaded liposomes (LipoLA) mainly depended on the LA loading concentration per single liposomes. Further study found that the LipoLA could fuse with the membranes of P. acnes and release the carried LA directly into the bacterial membranes, thereby killing the bacteria effectively. Since LA is a natural compound that is the main acid in coconut oil and also resides in human breast milk and liposomes have been successfully and widely applied as a drug delivery vehicle in the clinic, the LipoLA developed in this work holds great potential of becoming an innate, safe and effective therapeutic medication for acne vulgaris and other P. acnes associated diseases.     

Endocytosis of Streptococcus pneumoniae via the polymeric immunoglobulin receptor of epithelial cells relies on clathrin and caveolin dependent mechanisms

Colonization of Streptococcus pneumoniae (pneumococci) is a prerequisite for bacterial dissemination and their capability to enter the bloodstream. Pneumococci have evolved various successful strategies to colonize the mucosal epithelial barrier of humans. A pivotal mechanism of host cell invasion implicated with invasive diseases is promoted by the interaction of pneumococcal PspC with the polymeric Ig-receptor (pIgR). However, the mechanism(s) of pneumococcal endocytosis and the intracellular route of pneumococci upon uptake by the PspC–pIgR-interaction are not known. Here, we demonstrate by using a combination of pharmacological inhibitors and genetics interference approaches the involvement of active dynamin-dependent caveolae and clathrin-coated vesicles for pneumococcal uptake via the PspC–pIgR mechanism. Depleting cholesterol from host cell membranes and disruption of lipid microdomains impaired pneumococcal internalization. Moreover, chemical inhibition of clathrin or functional inactivation of dynamin, caveolae or clathrin by RNA interference significantly affected pneumococcal internalization suggesting that clathrin-mediated endocytosis (CME) and caveolae are involved in the bacterial uptake process. Confocal fluorescence microscopy of pIgR-expressing epithelial cells infected with pneumococci or heterologous Lactococcus lactis expressing PspC demonstrated bacterial co-localization with fluorescent-tagged clathrin and early as well as recycling or late endosomal markers such as Lamp1, Rab5, Rab4, and Rab7, respectively. In conclusion these data suggest that PspC-promoted uptake is mediated by both CME and caveolae. After endocytosis pneumococci are routed via the endocytic pathway into early endosomes and are then sorted into recycling or late endosomes, which can result in pneumococcal killing in phagolysosomes or transcytosis via recycling endosomes.     

Gold nanoparticles with a monolayer of doxorubicin-conjugated amphiphilic block copolymer for tumor-targeted drug delivery

Gold (Au) nanoparticles (NPs) stabilized with a monolayer of folate-conjugated poly(l-aspartate-doxorubicin)-b-poly(ethylene glycol) copolymer (Au-P(LA-DOX)-b-PEG-OH/FA) was synthesized as a tumor-targeted drug delivery carrier. The Au-P(LA-DOX)-b-PEG-OH/FA NPs consist of an Au core, a hydrophobic poly(l-aspartate-doxorubicin) (P(LA-DOX)) inner shell, and a hydrophilic poly(ethylene glycol) and folate-conjugated poly(ethylene glycol) outer shell (PEG-OH/FA). The anticancer drug, doxorubicin (DOX), was covalently conjugated onto the hydrophobic inner shell by acid-cleavable hydrazone linkage. The DOX loading level was determined to be 17 wt%. The Au-P(LA-DOX)-b-PEG-OH/FA NPs formed stable unimolecular micelles in aqueous solution. The size of the Au-P(LA-DOX)-b-PEG-OH/FA micelles were determined as 24–52 and 10–25 nm by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The conjugated DOX was released from the Au-P(LA-DOX)-b-PEG-OH/FA micelles much more rapidly at pH 5.3 and 6.6 than at pH 7.4, which is a desirable characteristic for tumor-targeted drug delivery. Cellular uptake of the Au-P(LA-DOX)-b-PEG-OH/FA micelles facilitated by the folate-receptor-mediated endocytosis process was higher than that of the micelles without folate. This was consistent with the higher cytotoxicity observed with the Au-P(LA-DOX)-b-PEG-OH/FA micelles against the 4T1 mouse mammary carcinoma cell line. These results suggest that Au-P(LA-DOX)-b-PEG-OH/FA NPs could be used as a carrier with pH-triggered drug releasing properties for tumor-targeted drug delivery.