Daya D. Verma

Atp-loaded liposomes for treatment of myocardial ischemia

A major obstacle to drug therapy for treatment of potential myocardial infarction is the limited access to the ischemic myocardium by drugs in an active form. Encouraging results have been reported with liposomes loaded with ATP in a variety of in vitro and in vivo models. We describe methods for optimized encapsulation of ATP in liposomes, enhancement of their effectiveness by increasing circulation time, and targeting of injured myocardial cells with surface attached antimyosin. In isolated ischemic rat hearts, ATP-loaded liposomes and ATP-loaded immunoliposomes effectively protected myocardium from ischemia/reperfusion damage as measured by systolic and diastolic functional improvements. In vivo, in rabbits with induced localized myocardial ischemia, liposomal encapsulation of ATP significantly diminished the proportion of ventricular muscle at risk that was irreversibly damaged during reperfusion. Therefore, ATP encapsulated in liposomes can provide an effective exogenous source for in vivo application which can protect ischemically damaged hearts.

ATP-loaded immunoliposomes specific for cardiac myosin provide improved protection of the mechanical functions of myocardium from global ischemia in an isolated rat heart model

Earlier demonstrated cardio-protection by ATP-loaded liposomes (ATP-L) was further improved by attachment of cardiac myosin-specific monoclonal 2G4 antibody onto the surface of ATP-L. ATP-IL were infused for 1 min duration before starting the global ischemia for 25 min followed by reperfusion for 30 min in an isolated rat heart. The left ventricular developed pressure at the end of reperfusion in ATP-IL group significantly recovered to above 80% of the baseline compared to ca 25% in the Krebs-Henseleit (KH) buffer, ca 60% in the IL, and ca 70% in the ATP-L treated groups. At the end of the reperfusion, left ventricular end diastolic pressure significantly reduced to 15 ± 2 mmHg in ATP-IL group compared to 59 ± 6 mmHg in the KH buffer, 31 ± 4 mmHg in the IL and 23 ± 3 mmHg in the ATP-L controls. The extent of preservation depended on the amount of the antibody present on the surface of the ATP-IL.

ATP-Loaded Liposomes for Targeted Treatment in Models of Myocardial Ischemia

ATP cannot be effectively delivered to most tissues including the ischemic myocardium without protection from degradation by plasma endonucleotidases. However, it has been established that ATP can be delivered to various tissues by its encapsulation within liposomal preparations. We describe here, the materials needed and methods used to optimize the encapsulation of ATP in liposomes, enhance their effectiveness by increasing their circulation time and target injured myocardial cells with liposomal surface anti-myosin antibody. Additionally, we outline methods for ex vivo studies of these ATP liposomal preparations in an isolated ischemic rat heart model and for in vivo studies of rabbits with an induced myocardial infarction. The expectation is that these methods will provide a basis for continued studies of effective ways to deliver energy substrates to the ischemic myocardium.

Confocal Microscopy for Visualization of Skin Penetration

Confocal microscopic techniques have been around for over 50 years. However, only in the last two decades has there been a truly widespread use of this technology in life sciences. Today there are different confocal techniques available and at the heart of all these techniques is the ability to differentiate between the light originating from different planes of the specimen. This ability is quite important when studying thick biological specimen such as skin microscopically, otherwise a lot of the information can get lost in the form of blur. In addition, due to advances in material sciences and photonics, microscopes are able to image in vitro as well as in vivo and have allowed us to couple microscopes with spectroscopic techniques which provide valuable information the in identification and characterization of structures. In this book chapter, we discuss basic principles of confocal laser scanning microscopy, 2-photon fluorescence microscopy, confocal Raman microscopy, coherent Raman microscopy, and their applications in understanding the skin penetration of various substances.

Invasomes: Vesicles for Enhanced Skin Delivery of Drugs

This chapter describes invasomes, which represent novel vesicular carriers for enhanced skin delivery. Invasomes are composed of unsaturated phospholipids, small amounts of ethanol and terpenes, and water. Different penetration studies performed in vitro in human skin will be represented in order to show the penetration-enhancing ability of invasomes. The first used invasomes or standard invasomes contained a terpene mixture, composed of cineole:citral:d-limonene = 45:45:10 v/v and were shown to be more efficient in delivering highly lipophilic and hydrophobic drugs into the skin, such as cyclosporine A (CsA) and temoporfin (mTHPC), compared to conventional liposomes and the ethanolic solution of drugs. It was also shown that invasomes significantly increased the permeation of hydrophilic drugs, like calcein and carboxyfluorescein, compared to conventional and deformable liposomes. Furthermore, also other terpene mixtures and single terpenes can be used to formulate invasomes, and dependent on the added terpene or terpene mixture, invasomes may enhance or retard the drug penetration into the skin compared to liposomes without terpenes. The presence of 1 % w/v citral, 1 % w/v cineole, or 1 % w/v standard terpene mixture in invasomes resulted in the formation of highly effective skin delivery systems for mTHPC, especially in the case of invasomes with 1 % w/v cineole providing high amounts of mTHPC not only in the SC but also in the deeper skin layers. As to the therapeutic effectiveness, the effectiveness of CsA invasomes containing 2 % w/v standard terpene mixture was confirmed in the treatment of alopecia areata in the Dundee Experimental Bald Rat (DEBR), while mTHPC invasomes containing 1 % w/v standard terpene mixture provided a slower tumor growth in mice bearing the subcutaneously located human colorectal HT29 carcinoma. The key factor for the high penetration-enhancing ability of invasomes is assumed to be their high fluidity confirmed by electron spin resonance, differential scanning calorimetry, and cryoelectron microscopy. However, as no direct correlation between the fluidity of invasomes and their ability to improve skin penetration of drugs was found, it is proposed that besides the fluidity, also other phenomena might be involved in the mechanism of the skin penetration enhancement induced by invasomes.