To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway.
In order to develop targeted pharmaceutical carriers additionally capable of responding certain local stimuli, such as decreased pH values in tumors or infarcts, targeted long-circulating PEGylated liposomes and PEG-phosphatidylethanolamine (PEG-PE)-based micelles have been prepared with several functions. First, they are capable of targeting a specific cell or organ by attaching the monoclonal antimyosin antibody 2G4 to their surface via pNP-PEG-PE moieties. Second, these liposomes and micelles were additionally modified with biotin or TAT peptide (TATp) moieties attached to the surface of the nanocarrier by using biotin-PE or TATp-PE or TATp-short PEG-PE derivatives. PEG-PE used for liposome surface modification or for micelle preparation was made degradable by inserting the pH-sensitive hydrazone bond between PEG and PE (PEG-Hz-PE). Under normal pH values, biotin and TATp functions on the surface of nanocarriers were "shielded" by long protecting PEG chains (pH-degradable PEG 2000 -PE or PEG 5000 -PE) or by even longer pNP-PEG-PE moieties used to attach antibodies to the nanocarrier (non-pH-degradable PEG 3400 -PE or PEG 5000 -PE). At pH 7.5-8.0, both liposomes and micelles demonstrated high specific binding with 2G4 antibody substrate, myosin, but very limited binding on an avidin column (biotin-containing nanocarriers) or internalization by NIH/3T3 or U-87 cells (TATp-containing nanocarriers). However, upon brief incubation (15-to-30 min) at lower pH values (pH 5.0-6.0) nanocarriers lost their protective PEG shell because of acidic hydrolysis of PEG-Hz-PE and acquired the ability to become strongly retained on avidin-column (biotin-containing nanocarriers) or effectively internalized by cells via TATp moieties (TATp-containing nanocarriers). We consider this result as the first step in the development of multifunctional stimuli-sensitive pharmaceutical nanocarriers.
A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot-labeled bone marrowderived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.Intravital microscopy has provided unprecedented molecular, cellular, anatomical and functional insight into tumor biology and response to treatment 1 . This technique captures fluorescence from molecules that are injected into a host or expressed by cells 2,3 . Additionally, intrinsic signals such as second harmonic generation (SHG) emanating from collagen can be imaged using multiphoton microscopy 4,5 . Traditional fluorophores are prone to photobleaching, compromising the ability to image the same region repeatedly, and have relatively narrow excitation and broad emission spectra. Also, several excitation wavelengths may be required to excite all fluorophores and intrinsic signals, and overlapping emissions may obscure the delineation between multiple probes. Quantum dots, colloidal semiconductor nanocrystals 6 , have the potential to overcome these limitations: they are photostable, tunable to a desired narrow emission spectrum, relatively insensitive to the wavelength of excitation Correspondence should be addressed to R.K.J. (E-mail: jain@steele.mgh.harvard.edu). Note: Supplementary information is available on the Nature Medicine website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. light, and are especially bright fluorophores 7 . Recent studies exploit these optical properties for imaging of cells 8 or whole tumors 9 . The ability of quantum dots to show crucial information at the length scale between these two extremes has yet to be established 10 . Here, we present studies that highlight the synergy of quantum dots and multiphoton intravital microscopy for tumor pathophysiology studies: differentiating tumor vessels from both perivascular cells and matrix, assaying the ability of microparticles to access the tumor, and monitoring the trafficking of precursor cells. NIH Public Access RESULTS Customizing quantum dot emissionBecause quantum dot emissions are tunable by both size and chemical composition 6 , we prepared ...
We have attempted to simplify the procedure for coupling various ligands to distal ends of liposome-grafted polyethylene glycol (PEG) chains and to make it applicable for single-step binding of a large variety of a primary amino group-containing substances, including proteins and small molecules. With this in mind, we have introduced a new amphiphilic PEG derivative, p-nitrophenylcarbonyl-PEG-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (pNP-PEG-DOPE), synthesized by reaction of DOPE with excess of bis(p-nitrophenylcarbonyl)-PEG in a chloroform/triethylamine mixture. pNP-PEG-DOPE readily incorporates into liposomes via its PE residue, and easily binds primary amino group-containing ligands via its water-exposed pNP groups, forming stable and non-toxic urethane (carbamate) bonds. The reaction between the pNP group and the ligand amino group proceeds easily and quantitatively at pH around 8.0, and remaining free pNP groups are promptly eliminated by spontaneous hydrolysis. Therefore, pNP-PEG-DOPE could serve as a very convenient tool for protein attachment to the distal ends of liposome-grafted PEG chains. To investigate the applicability of the suggested protocol for the preparation of long-circulating targeted liposomes, we have coupled several proteins, such as concanavalin A (ConA), wheat germ agglutinin (WGA), avidin, monoclonal antimyosin antibody 2G4 (mon2G4), and monoclonal antinucleosome antibody 2C5 (mon2C5) to PEG-liposomes via terminal pNP groups and studied whether the specific activity of these immobilized proteins is preserved. The method permits the binding of several dozens protein molecules per single 200 nm liposome. All bound proteins completely preserve their specific activity. Lectin-liposomes are agglutinated by the appropriate polyvalent substrates (mannan for ConA-liposomes and glycophorin for WGA-liposomes); avidin-liposomes specifically bind with biotin-agarose; antibody-liposomes demonstrate high specific binding to the substrate monolayer both in the direct binding assay and in ELISA. A comparison of the suggested method with the method of direct membrane incorporation was made. The effect of the concentration of liposome-grafted PEG on the preservation of specific protein activity in different coupling protocols was also investigated. It was also shown that pNP-PEG-DOPE-liposomes with and without attached ligands demonstrate increased stability in mouse serum.
Purpose:To determine if oxidative and nitrative stress and/or apoptosis contribute to increased coagulation when combining radiofrequency (RF) ablation with liposomal doxorubicin. Materials and Methods:Animal care committee approval was obtained. R3230 mammary adenocarcinomas in Fischer rats were treated with either RF ablation ( n = 43), 1 mg of intravenously injected liposomal doxorubicin ( n = 26), or combined therapy ( n = 30) and were compared with control subjects ( n = 11). A subset of animals receiving combination therapy ( n = 24) were treated in the presence or absence of N -acetylcysteine (NAC) administered 24 hours and 1 hour before RF ablation. Tumors were analyzed 2 minutes to 72 hours after treatment to determine the temporal range of response by using immunohistochemical staining of the apoptosis marker cleaved caspase-3, phosphorylated g H2AX , and Results:By 4 hours after RF ablation alone, a 0.48-mm 6 0.13 (standard deviation ) peripheral band with 57.0% 6 7.3 cleaved caspase-3 positive cells was noted at the ablation margin, whereas a 0.73-mm 6 0.18 band with 77.7% 6 6.3 positivity was seen for combination therapy ( P , .03 for both comparisons). Combination therapy caused increased and earlier staining for 4-HNE-modifi ed proteins, 8-OHdG, NT, and g H2AX with colocalization to cleaved caspase-3 staining. A rim of increased HSP70 was identifi ed peripheral to the area of cleaved caspase-3. Parameters of oxidative and nitrative stress were signifi cantly inhibited by NAC 1 hour following RF ablation, resulting in decreased cleaved caspase-3 positivity (0.28-mm 6 0.09 band of 25.9% 6 7.4 positivity vs 0.59-mm 6 0.11 band of 62.9% 6 6.0 positivity, P , .001 for both comparisons). Conclusion:Combining RF ablation with liposomal doxorubicin increases cell injury and apoptosis in the zone of increased coagulation by using a mechanism that involves oxidative and nitrative stress that leads to accelerated apoptosis.q RSNA, 2010
PurposeRadiofrequency thermal ablation (RFA) of hepatic and renal tumors can be accompanied by non-desired tumorigenesis in residual, untreated tumor. Here, we studied the use of micelle-encapsulated siRNA to suppress IL-6-mediated local and systemic secondary effects of RFA.MethodsWe compared standardized hepatic or renal RFA (laparotomy, 1 cm active tip at 70±2°C for 5 min) and sham procedures without and with administration of 150nm micelle-like nanoparticle (MNP) anti-IL6 siRNA (DOPE-PEI conjugates, single IP dose 15 min post-RFA, C57Bl mouse:3.5 ug/100ml, Fisher 344 rat: 20ug/200ul), RFA/scrambled siRNA, and RFA/empty MNPs. Outcome measures included: local periablational cellular infiltration (α-SMA+ stellate cells), regional hepatocyte proliferation, serum/tissue IL-6 and VEGF levels at 6-72hr, and distant tumor growth, tumor proliferation (Ki-67) and microvascular density (MVD, CD34) in subcutaneous R3230 and MATBIII breast adenocarcinoma models at 7 days.ResultsFor liver RFA, adjuvant MNP anti-IL6 siRNA reduced RFA-induced increases in tissue IL-6 levels, α-SMA+ stellate cell infiltration, and regional hepatocyte proliferation to baseline (p<0.04, all comparisons). Moreover, adjuvant MNP anti-IL6- siRNA suppressed increased distant tumor growth and Ki-67 observed in R3230 and MATBIII tumors post hepatic RFA (p<0.01). Anti-IL6 siRNA also reduced RFA-induced elevation in VEGF and tumor MVD (p<0.01). Likewise, renal RFA-induced increases in serum IL-6 levels and distant R3230 tumor growth was suppressed with anti-IL6 siRNA (p<0.01).ConclusionsAdjuvant nanoparticle-encapsulated siRNA against IL-6 can be used to modulate local and regional effects of hepatic RFA to block potential unwanted pro-oncogenic effects of hepatic or renal RFA on distant tumor.
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