Synthesis of a Gemcitabine Prodrug for Remote Loading into Liposomes and Improved Therapeutic Effect

The chemotherapeutic gemcitabine was actively and stably loaded into lipid nanoparticles through the formation of a prodrug. Gemcitabine was chemically modified to increase the lipophilicity and introduce a weak base moiety for remote loading. Several derivatives were synthesized and screened for their potential to be good liposomal drug candidates for remote loading by studying their solubility, stability, cytotoxicity, and loading efficiency. Two morpholino derivatives of GEM (22 and 23) were chosen as the preferred prodrugs for this purpose as they possessed the best loading efficiencies (100% for drug-to-lipid ratio of 0.36 w/w). This is a considerable improvement over a passive loading strategy where typical loading efficiencies are on the order of ∼10-20% for a drug-to-lipid ratio of ∼0.01. Liposomes loaded with these two prodrugs were studied in an s.c. tumor model in vivo and showed improved therapeutic effect over free GEM (∼2-fold) and saline control (8- to 10-fold). This work demonstrates how chemical modification of a known hydrophilic drug can lead to improved loading, stability, and drug delivery in vivo.</p

Thermosensitive liposomes for the delivery of gemcitabine and oxaliplatin to tumors

The majority of ultrafast temperature sensitive liposome (uTSL) formulations reported in the literature deliver the highly membrane permeable drug, doxorubicin (DOX). Here we report on the study of the uTSL formulation, HaT (Heat activated cytoToxic, composed of the phospholipid DPPC and the surfactant Brij78) loaded with the water-soluble, but poorly membrane permeable anticancer drugs, gemcitabine (GEM) and oxaliplatin (OXA). The HaT formulation displayed ultrafast release of these drugs in response to temperature, whereas attempts with LTSL (Lyso-lipid Temperature Sensitive Liposome, composed of DPPC, MSPC, and DSPE-PEG) were unsuccessful. HaT-GEM and HaT-OXA both released &gt;80% of the encapsulated drug within 2 min at 40-42°C, with &lt;5% drug leakage at 37°C after 30 min in serum. The pharmacokinetic profile of both drugs was improved by formulating with HaT relative to the free drug, with clearance reduced by 50-fold for GEM and 3-fold for OXA. HaT-GEM and HaT-OXA both displayed improved drug uptake in the heated tumor relative to the unheated tumor (by 9-fold and 3-fold, respectively). In particular, HaT-GEM showed 25-fold improved delivery to the heated tumor relative to free GEM and significantly enhanced antitumor efficacy with complete tumor regression after a single dose of HaT-GEM. These data suggest that uTSL technology can also be used to deliver nonmembrane permeable drugs via an intravascular ultrafast release mechanism to great effect.</p

A Study of Liposomal Formulations to Improve the Delivery of Aquated Cisplatin to a Multidrug Resistant Tumor

Purpose: This study was aimed at exploring the use of liposomes to deliver aquated cisplatin (ACP), a metabolite of CDDP, with increased potency and toxicity. Three liposomal formulations were compared for delivery of ACP to a multidrug resistant tumor. Methods: Three different liposomes (DMPC, DPPC and DSPC as the main lipid components) were loaded with ACP by the thin-film hydration method. In vitro drug release was assessed over 72 h at 37°C in PBS. The pharmacokinetics of free CDDP and the three ACP liposomes was determined using ICP-AES and their efficacy against EMT6-AR1 multidrug resistant murine breast tumor was compared. Results: The DSPC formulation, composed of a C18 acyl chain lipid, exhibited the slowest drug release (~2%) after 72 h at 37°C, compared to the other two formulations with decreased carbon chain lengths (C16 and C14; 7 and 25% release respectively). The pharmacokinetic profile was improved with all liposomal formulations relative to free CDDP, with clearance reduced by 500-fold for DSPC, 200-fold for DPPC and 130-fold for DMPC. The DSPC formulation displayed the highest drug accumulation in the tumor with 2-fold, 3-fold and 100-fold increases compared to DPPC, DMPC and free CDDP respectively. The DSPC formulation significantly inhibited the EMT6-AR1 tumor growth by ~90%, while the other formulations displayed no statistically significant improved activity compared to saline. Conclusion: These results suggest that the DSPC liposomal formulation is a promising formulation for MDR tumor therapy over DMPC and DPPC formulations and free drug.</p

Synthesis of a Gemcitabine Prodrug for Remote Loading into Liposomes and Improved Therapeutic Effect

The chemotherapeutic gemcitabine was actively and stably loaded into lipid nanoparticles through the formation of a prodrug. Gemcitabine was chemically modified to increase the lipophilicity and introduce a weak base moiety for remote loading. Several derivatives were synthesized and screened for their potential to be good liposomal drug candidates for remote loading by studying their solubility, stability, cytotoxicity, and loading efficiency. Two morpholino derivatives of GEM (<b>22</b> and <b>23</b>) were chosen as the preferred prodrugs for this purpose as they possessed the best loading efficiencies (100% for drug-to-lipid ratio of 0.36 w/w). This is a considerable improvement over a passive loading strategy where typical loading efficiencies are on the order of ∼10–20% for a drug-to-lipid ratio of ∼0.01. Liposomes loaded with these two prodrugs were studied in an s.c. tumor model in vivo and showed improved therapeutic effect over free GEM (∼2-fold) and saline control (8- to 10-fold). This work demonstrates how chemical modification of a known hydrophilic drug can lead to improved loading, stability, and drug delivery in vivo

Correction: Photoacoustic Imaging of Cancer Treatment Response: Early Detection of Therapeutic Effect from Thermosensitive Liposomes.

[This corrects the article DOI: 10.1371/journal.pone.0165345.]

Thermosensitive Liposomes for the Delivery of Gemcitabine and Oxaliplatin to Tumors

The majority of ultrafast temperature sensitive liposome (uTSL) formulations reported in the literature deliver the highly membrane permeable drug, doxorubicin (DOX). Here we report on the study of the uTSL formulation, HaT (<b>H</b>eat <b>a</b>ctivated cyto<b>T</b>oxic, composed of the phospholipid DPPC and the surfactant Brij78) loaded with the water-soluble, but poorly membrane permeable anticancer drugs, gemcitabine (GEM) and oxaliplatin (OXA). The HaT formulation displayed ultrafast release of these drugs in response to temperature, whereas attempts with LTSL (<b>L</b>yso-lipid <b>T</b>emperature <b>S</b>ensitive <b>L</b>iposome, composed of DPPC, MSPC, and DSPE-PEG) were unsuccessful. HaT-GEM and HaT-OXA both released >80% of the encapsulated drug within 2 min at 40–42 °C, with <5% drug leakage at 37 °C after 30 min in serum. The pharmacokinetic profile of both drugs was improved by formulating with HaT relative to the free drug, with clearance reduced by 50-fold for GEM and 3-fold for OXA. HaT-GEM and HaT-OXA both displayed improved drug uptake in the heated tumor relative to the unheated tumor (by 9-fold and 3-fold, respectively). In particular, HaT-GEM showed 25-fold improved delivery to the heated tumor relative to free GEM and significantly enhanced antitumor efficacy with complete tumor regression after a single dose of HaT-GEM. These data suggest that uTSL technology can also be used to deliver nonmembrane permeable drugs via an intravascular ultrafast release mechanism to great effect

Photoacoustic signal characterization of cancer treatment response: Correlation with changes in tumor oxygenation

Frequency analysis of the photoacoustic radiofrequency signals and oxygen saturation estimates were used to monitor the in-vivo response of a novel, thermosensitive liposome treatment. The liposome encapsulated doxorubicin (HaT-DOX) releasing it rapidly (<20 s) when the tumor was exposed to mild hyperthermia (43 °C). Photoacoustic imaging (VevoLAZR, 750/850 nm, 40 MHz) of EMT-6 breast cancer tumors was performed 30 min pre- and post-treatment and up to 7 days post-treatment (at 2/5/24 h timepoints). HaT-DOX-treatment responders exhibited on average a 22% drop in oxygen saturation 2 h post-treatment and a decrease (45% at 750 nm and 73% at 850 nm) in the slope of the normalized PA frequency spectra. The spectral slope parameter correlated with treatment-induced hemorrhaging which increased the optical absorber effective size via interstitial red blood cell leakage. Combining frequency analysis and oxygen saturation estimates differentiated treatment responders from non-responders/control animals by probing the treatment-induced structural changes of blood vessel

Photoacoustic radiofrequency spectroscopy for monitoring cancer treatment response

Frequency analysis of the photoacoustic (PA) signals was combined with functional PA estimations of tumor oxygenation to monitor the in-vivo response of a temperature sensitive liposome treatment. Mouse breast cancer tumors were imaged using the Vevo LAZR system (Fujifilm VisualSonics, Toronto, Canada) before treatment and 30 min/2h/5h/24h/7d post-treatment. Treatment consisted of an injection of either the liposome (HaT-DOX) or saline followed by heating of the tumour in a water bath for 1 hour at 43°C. The tumor oxygenation and the slope of the normalise power spectrum decreased by 22% and 45% at 750 nm, respectively as early as 30 minutes post-treatment. The combined analysis was able to differentiate responding mice from their non-responding counterparts and the control group. These results suggest that probing both structural (spectral slope) and functional (oxygen saturation) changes that occur during cancer treatment can aid in identifying treatment response.</p

Longitudinal monitoring of oxygen saturation with photoacoustic imaging

Assessing the response of cancer treatments non-invasively on an individual (or patient) basis has the potential to impact cancer treatment. The need for a modality that provides multifaceted information about the structural and metabolic changes that occur within tumors is important, especially because of the vasculature and the role it plays in tumor growth. One such modality capable of imaging the anatomy and functionality of vasculature is photoacoustic (PA) imaging. In this study, in vivo PA imaging and estimation of oxygen saturation (SO2) was performed longitudinally to monitor the efficacy of thermosensitive liposome delivery vehicles. By mapping the distribution of oxygen in 12 animals treated with doxorubicin-loaded thermosensitive liposomes, early SO2 changes (within hours) were predictive of treatment success as assessed by volumetric tumor growth after 2 weeks. Twelve saline-treated animals showed no observable, early changes in SO2 and displayed larger intertumoral variability in oxygen distribution compared to the treated group. The longitudinal variations in estimated tumor oxygenation, particularly in the first 5 hours post treatment, demonstrate the unique sensitivity of PA imaging to monitor these functional changes in tumor vasculature.</p