Investigation of the Acoustic Vaporization Threshold of Lipid-Coated Perfluorobutane Nanodroplets Using Both High-Speed Optical Imaging and Acoustic Methods.

A combination of ultrahigh-speed optical imaging (5 × 106 frames/s), B-mode ultrasound and passive cavitation detection was used to study the vaporization process and determine both the acoustic droplet vaporization (ADV) and inertial cavitation (IC) thresholds of phospholipid-coated perfluorobutane nanodroplets (PFB NDs, diameter = 237 ± 16 nm). PFB NDs have not previously been studied with ultrahigh-speed imaging and were observed to form individual microbubbles (1-10 µm) within two to three cycles and subsequently larger bubble clusters (10-50 µm). The ADV and IC thresholds did not statistically significantly differ and decreased with increasing pulse length (20-20,000 cycles), pulse repetition frequency (1-100 Hz), concentration (108-1010 NDs/mL), temperature (20°C-45°C) and decreasing frequency (1.5-0.5 MHz). Overall, the results indicate that at frequencies of 0.5, 1.0 and 1.5 MHz, PFB NDs can be vaporized at moderate peak negative pressures (<2.0 MPa), pulse lengths and pulse repetition frequencies. This finding is encouraging for the use of PFB NDs as cavitation agents, as these conditions are comparable to those required to achieve therapeutic effects with microbubbles, unlike those reported for higher-boiling-point NDs. The differences between the optically and acoustically determined ADV thresholds, however, suggest that application-specific thresholds should be defined according to the biological/therapeutic effect of interest.

Tailoring the size of ultrasound responsive lipid-shelled nanodroplets by varying production parameters and environmental conditions.

Liquid perfluorocarbon nanodroplets (NDs) are an attractive alternative to microbubbles (MBs) for ultrasound-mediated therapeutic and diagnostic applications. ND size and size distribution have a strong influence on their behaviour in vivo, including extravasation efficiency, circulation time, and response to ultrasound stimulation. Thus, it is desirable to identify ways to tailor the ND size and size distribution during manufacturing. In this study phospholipid-coated NDs, comprising a perfluoro-n-pentane (PFP) core stabilised by a DSPC/PEG40s (1,2-distearoyl-sn-glycero-3-phosphocholine and polyoxyethylene(40)stearate, 9:1 molar ratio) shell, were produced in phosphate-buffered saline (PBS) by sonication. The effect of the following production-related parameters on ND size was investigated: PFP concentration, power and duration of sonication, and incorporation of a lipophilic fluorescent dye. ND stability was also assessed at both 4 °C and 37 °C. When a sonication pulse of 6 s and 15% duty cycle was employed, increasing the volumetric concentration of PFP from 5% to 15% v/v in PBS resulted in an increase in ND diameter from 215.8 ± 16.8 nm to 408.9 ± 171.2 nm. An increase in the intensity of sonication from 48 to 72 W (with 10% PFP v/v in PBS) led to a decrease in ND size from 354.6 ± 127.2 nm to 315.0 ± 100.5 nm. Increasing the sonication time from 20 s to 40 s (using a pulsed sonication with 30% duty cycle) did not result in a significant change in ND size (in the range 278-314 nm); however, when it was increased to 60 s, the average ND diameter reduced to 249.7 ± 9.7 nm, which also presented a significantly lower standard deviation compared to the other experimental conditions investigated (i.e., 9.7 nm vs. > 49.4 nm). The addition of the fluorescent dye DiI at different molar ratios did not affect the ND size distribution. NDs were stable at 4 °C for up to 6 days and at 37 °C for up to 110 min; however, some evidence of ND-to-MB phase transition was observed after 40 min at 37 °C. Finally, phase transition of NDs into MBs was demonstrated using a tissue-mimicking flow phantom under therapeutic ultrasound exposure conditions (ultrasound frequency: 0.5 MHz, acoustic pressure: 2-4 MPa, and pulse repetition frequency: 100 Hz).

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.

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.].

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

Imaging methods capable of indicating the potential for success of an individualized treatment course, during or immediately following the treatment, could improve therapeutic outcomes. Temperature Sensitive Liposomes (TSLs) provide an effective way to deliver chemotherapeutics to a localized tumoral area heated to mild-hyperthermia (HT). The high drug levels reached in the tumor vasculature lead to increased tumor regression via the cascade of events during and immediately following treatment. For a TSL carrying doxorubicin (DOX) these include the rapid and intense exposure of endothelial cells to high drug concentrations, hemorrhage, blood coagulation and vascular shutdown. In this study, ultrasound-guided photoacoustic imaging was used to probe the changes to tumors following treatment with the TSL, HaT-DOX (Heat activated cytoToxic). Levels of oxygen saturation (sO2) were studied in a longitudinal manner, from 30 min pre-treatment to 7 days post-treatment. The efficacious treatments of HT-HaT-DOX were shown to induce a significant drop in sO2 (>10%) as early as 30 min post-treatment that led to tumor regression (in 90% of cases); HT-Saline and non-efficacious HT-HaT-DOX (10% of cases) treatments did not show any significant change in sO2 at these timepoints. The changes in sO2 were further corroborated with histological data, using the vascular and perfusion markers CD31 and FITC-lectin. These results allowed us to further surmise a plausible mechanism of the cellular events taking place in the TSL treated tumor regions over the first 24 hours post-treatment. The potential for using photoacoustic imaging to measure tumor sO2 as a surrogate prognostic marker for predicting therapeutic outcome with a TSL treatment is demonstrated.

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.

Synthesis of a Cytotoxic Amanitin for Biorthogonal Conjugation.

Alpha-amanitin is an exceedingly toxic, naturally occurring, bicyclic octapeptide that inhibits RNA polymerase and results in cellular and organismal death. Here we report the straightforward synthesis of an amanitin analogue that exhibited near-native toxicity. A pendant alkyne was readily installed to enable copper-catalyzed alkyne-azide cycloaddition (CuAAC) to azido-rhodamine and two azide-bearing versions of the RGD peptide. The fluorescent toxin analogue entered cells and provoked morphological changes consistent with cell death. The latter two conjugates are as toxic as the parent alkyne precursor, which demonstrates that conjugation does not diminish toxicity. In addition, we showed that toxicity depends on a single diastereomer of the unnatural amino acid, dihydroxyisoleucine (DHIle), at position 3. The convenient synthesis of a heptapeptide precursor now provides access to bioactive amanitin analogues that may be readily conjugated to biomolecules of interest.

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.

Recent progress in the development of polysaccharide conjugates of docetaxel and paclitaxel.

UNLABELLED: Taxanes are one of the most potent and broadest spectrum chemotherapeutics used clinically, but also induce significant side effects. Different strategies have been developed to produce a safer taxane formulation. Development of polysaccharide drug conjugates has increased in the recent years because of the demonstrated biocompatibility, biodegradability, safety, and low cost of the biopolymers. This review focuses on polysaccharide-taxane conjugates and provides an overview on various conjugation strategies and their effect on the efficacy. Detailed analyses on the designing factors of an effective polysaccharide-drug conjugate are provided with a discussion on the future direction of this field. For further resources related to this article, please visit the WIREs website. CONFLICT OF INTEREST: The authors have declared no conflicts of interest for this article.

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 >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.

Hyperthermia-induced drug targeting.

INTRODUCTION: Specific delivery of a drug to a target site is a major goal of drug delivery research. Using temperature-sensitive liposomes (TSLs) is one way to achieve this; the liposome acts as a protective carrier, allowing increased drug to flow through the bloodstream by minimizing clearance and non-specific uptake. On reaching microvessels within a heated tumor, the drug is released and quickly penetrates. A major advance in the field is ThermoDox® (Celsion), demonstrating significant improvements to the drug release rates and drug uptake in heated tumors (∼ 41°C). Most recently, magnetic resonance-guided focused ultrasound (MRgFUS) has been combined with TSL drug delivery to provide localized chemotherapy with simultaneous quantification of drug release within the tumor. AREAS COVERED: In this article the field of hyperthermia-induced drug delivery is discussed, with an emphasis on the development of TSLs and their combination with hyperthermia (both mild and ablative) in cancer therapy. State-of-the-art image-guided heating technologies used with this combination strategy will also be presented, with examples of real-time monitoring of drug delivery and prediction of efficacy. EXPERT OPINION: The specific delivery of drugs by combining hyperthermia with TSLs is showing great promise in the clinic and its potential will be even greater as the use of image-guided focused ultrasound becomes more widespread - a technique capable of penetrating deep within the body to heat a specific area with improved control. In conjunction with this, it is anticipated that multifunctional TSLs will be a major topic of study in this field.

A thermosensitive liposome prepared with a Cu²âº gradient demonstrates improved pharmacokinetics, drug delivery and antitumor efficacy.

Here we report the development of an enhanced thermosensitive formulation composed of DPPC and Brij78, loaded with doxorubicin (DOX) using a Cu²âº gradient and post-inserted with an additional amount of Brij78. This optimal formulation (HaT-II: Hyperthermia-activated cytoToxic) displayed significantly improved stability in serum at 37 °C, and enhanced drug release rates at 41-42 °C, compared to LTSL (lyso-lipid temperature sensitive liposomes, DPPC/MSPC/DSPE-PEG2000=86/10/4, pH gradient drug loading). HaT-II released 100% DOX within 15-40s at 40-42 °C, with only 5% drug leakage at 37 °C after 30 min in serum, while LTSL lost 30% of its drug content at 37 °C and exhibited ~2-fold decreased release rate constants at 41-42 °C under the same conditions. The pharmacokinetics of DOX was significantly improved in non-heated HaT-II treated healthy mice with 2.5-fold increased area under the curve and 2-fold prolonged circulation half life compared to LTSL. This led to 2-fold improved drug delivery to the heated tumor by HaT-II (~20% injected dose/g tissue), relative to LTSL and significantly enhanced antitumor efficacy with complete inhibition of tumor growth after a single dose of HaT-II. Finally, HaT-II exhibited little toxicity in mice, inducing no body weight loss and no abnormality in the blood chemistry (10 mg DOX/kg).

Stereoselective synthesis of brevianamide E.

The hydroxypyrroloindolenine (Hpi) motif forms the fundamental core of the pentacyclic natural product, brevianamide E, the concise stereoselective synthesis of which, via oxidative cyclization, is described.

MRI monitoring of intratumoral drug delivery and prediction of the therapeutic effect with a multifunctional thermosensitive liposome.

Non-invasive in vivo imaging of drug distribution enables real-time monitoring and prediction of therapeutic responses to treatment. We have developed a thermosensitive liposomal formulation (HaT: Hyperthermia-activated-cytoToxic) consisting of DPPC and Brij78, a formulation that enhanced drug delivery compared to the lyso-lipid temperature sensitive liposomes (LTSL). Here we report the development of a multifunctional HaT liposome co-encapsulating Gd-DTPA (an MRI probe) and doxorubicin (DOX), which simultaneously releases and reports on drug delivery in a locally heated tumor. The temperature-dependent release profiles of DOX from HaT were closely related to the change in the MR T(1) relaxation time, in which DOX was 100% released at 40-42 °C in 3 min, accompanied by a 60% reduction in T(1). By T(1) relaxometry analysis, no Gd-DTPA leakage was detected in 30 min at 30-37 °C. In the in vivo study, DOX uptake in the tumor was quantitatively correlated with T(1) response (R(2) = 0.98) and the patterns of the T(1) image and the intratumoral DOX uptake were matched, in which both signals were predominantly detected in the highly perfused tumor periphery. Finally, the extent of T(1) relaxation enhancement in the heated tumor successfully predicted the antitumor efficacy in a standard pharmacological response model (R(2) = 0.98).

Synthesis, characterisation, and in vitro evaluation of Pro2-Ile3-S-deoxo-amaninamide and Pro2-D-allo-Ile3-S-deoxo-amaninamide: implications for structure-activity relationships in amanitin conformation and toxicity.

The amatoxins are a family of toxic bicyclic peptides that inhibit RNA polymerase II. Herein we discuss an improved synthesis of these compounds from easily obtainable amino acids by means of a solid-phase methodology. Interestingly, we obtained two products of the same mass following our final macrocyclisation, relating to a similar distribution of products described in some previous reports. One of these products was the desired amatoxin; Pro(2)-Ile(3)-S-deoxo-amaninamide 1 b. The other compound, after thorough investigation, was confirmed to be the epimer Pro(2)-D-allo-Ile(3)-S-deoxo-amaninamide 1 a, not an atropisomer structure as previously suggested in syntheses of related amanitin analogues. Crystallographic data of 1 a confirms the presence of a betaII-turn, rather than a betaI-turn common to the natural toxin and 1 b. This difference explains the large variation in CD spectra, although it seems to have relatively little effect on the bioactivity in vitro. These data provide new insights into the bicyclic amatoxin structure.

Intraannular Savige-Fontana reaction: one-step conversion of one class of monocyclic peptides into another class of bicyclic peptides.

Cyclisation and cross-linking strategies are important for the synthesis of cyclic and bicyclic peptides. These macrolactams are of great interest due to their increased biological activity compared to linear analogues. Herein, we describe the synthesis of a cyclic peptide containing an Hpi toxicophore, reminiscent of phakellistatins and omphalotins. The first intraannular cross-linking of such a peptide is then presented: using neat TFA to catalyse a Savige-Fontana tryptathionylation, the Hpi-containing peptide is converted to a bicyclic amatoxin analogue. As such, this methodology represents an efficient cyclisation method for cross-linking peptides and exposes a heretofore unrealised relationship between two different classes of peptide natural products. This finding increases the degree of potential chemical space for library generation.

Tryptathionine bridges in peptide synthesis.

The tryptathionine linkage is a crosslink formed between tryptophan and cysteine. This feature is characteristic of the bicyclic peptides: the phallotoxins and the amatoxins. These peptides both bind to protein folds of their respective targets (F-actin and RNA pol II, respectively) with extremely high affinities. Studies on these peptides have shown that the tryptathionine crosslink is essential for this binding affinity. Tryptathionines have been investigated for many years and several syntheses exist for their formation. In this review, we report on the various methodologies employed in tryptathionine synthesis, and discuss some of the advantages and disadvantages associated with each of them.

High yielding synthesis of 3a-hydroxypyrrolo[2,3-b]indoline dipeptide methyl esters: synthons for expedient introduction of the hydroxypyrroloindoline moiety into larger peptide-based natural products and for the creation of tryptathionine bridges.

This work describes a rapid and high yielding oxidation of 14 tryptophanylated amino acid methyl esters to the corresponding 3a-hydroxypyrrolo[2,3-b]indoline (Hpi) amino acids with generally facile separation of syn-cis and anti-cis diastereomers. Structural X-ray diffraction data are presented for both diastereomers of Tr-Hpi-Gly-OMe, which allow for a putative assignment of the other 13 pairs of diastereomers reported herein, based on correlations with 1H NMR chemical shifts. Selective and high yielding deprotection at either the N or C terminus is described, allowing the Hpi motif to be introduced efficiently into potential targets with minimal protecting group manipulation. Two tripeptides containing Hpi and cysteine were prepared and treated with acid in the Savige-Fontana reaction to produce a cyclic tryptathionine linkage, characteristic of both amatoxins and phallotoxins.

Synthesis and evaluation of a new non-fluorescent quencher in fluorogenic oligonucleotide probes for real-time PCR.

A non-fluorescent quencher, based on the diaminoanthraquinone Disperse Blue 3, has been incorporated into oligonucleotides at the 5'-end, the 3'-end and internally as a thymidine derivative. Fluorimetry and fluorogenic real-time PCR experiments demonstrate that the quencher is effective with a wide range of fluorescent dyes. The anthraquinone moiety increases the melting temperature of DNA duplexes, thus allowing shorter, more discriminatory probes to be used. The quencher has been used in Scorpion primers and TaqMan probes for human DNA sequence recognition and mutation detection.

Covalent Schiff base catalysis and turnover by a DNAzyme: a M2+ -independent AP-endonuclease mimic.

A DNAzyme, synthetically modified with both primary amines and imidazoles, is found to act as a M2+ -independent AP lyase-endonuclease. In the course of the cleavage reaction, this DNAzyme forms a covalent Schiff base intermediate with an abasic site on a complementary oligodeoxyribonucleotide. This intermediate, which is inferred from NaCNBH3 trapping as well as cyanide inhibition, does not evidently accumulate because the second step, dehydrophosphorylative elimination, is fast compared to Schiff base formation. The 5'-product that remains linked to the catalyst hydrolyzes slowly to regenerate free catalyst. The use of duly modified DNAzymes to perform Schiff base catalysis demonstrates the value of modified nucleotides for enhancing the catalytic repertoire of nucleic acids. This work suggests that DNAzymes will be capable of catalyzing aldol condensation reactions.