Laser Optofluidics in Fighting Multiple Drug Resistance

This chapter contains basic data about the microfluidic description of pendant droplets. Results are shown regarding the surface/interfacial tension measurements performed on water based solutions following the interaction with laser radiation. A synthesis is introduced of the main methods used to produce simple or complex droplets in different media. A method to evidence surface active products obtained after exposure of medicine solutions to laser radiation is presented. It consists in measuring in real time the dynamic interfacial tension at the interface between air and irradiated solution, when solution is in bulk form. The variation of dynamic interfacial tension is an indicator of the presence of laser produced amphiphilic molecules in solution. These results belong to series of reports dedicated to new methods used to fight multiple drug resistance developed by bacteria by decreasing the concentration of active compounds with bactericide effects. In line with microfluidic approach of droplets with μl volumes, surface tension measurements on DMSO-water mixtures containing a dye are presented.

This chapter presents a synthesis regarding the optical properties of pendant droplets in view of describing and understanding their interactions with laser and, more general, optical beams. The main methods used for pendant droplets investigation are described, from the optical point of view, at unresonant and resonant interaction with a laser beam focused or sent on it. The interaction is considered in the excitation scheme one laser pulse - one microvolumetric droplet in pendant position in air with typical volumes of the droplet in 1-15 μl range. In the unresonant interaction case the laser beam is not absorbed by droplet’s material(s). Beam propagation in droplet is made according to geometrical optics rules when the separation surface between two optical media (air and water, for instance) is spherical. Total reflection of laser beam within droplet at separating surface with respect to the air can be produced and this can give a particular brightness of an illuminated droplet. At resonant interaction, the beam is absorbed by droplet’s material(s) and typical phenomena may take place such as laser induced fluorescence (LIF) emission and Raman scattering. These effects are described here. LIF emitted by microdroplets of Rhodamine 6G solution in water is described and results about its amplification in the droplet considered as a spherical optical resonator are shown to the limit of obtaining lasing effects. Raman spectra of dimethyl sulfoxide and ultrapure water microvolumetric droplets are shown and a comparison between Raman beams emitted by microdroplets and bulky samples is made.

Bubble and drop profile analysis tensiometry is one of the most versatile tools for the characterisation of liquid interfaces. This technique provides the time dependence of surface and interfacial tension in a brought time range. The obtained interfacial tensions are well suited for a quantitative characterisation of adsorption layers of surfactants, proteins, polymers, and of their mixtures at liquid interfaces. Instruments equipped with an accurate dosing system allow experiments under well controlled conditions, such as constant drop/bubble volume or surface area. Also preprogramed changes are feasible, for example drop volume oscillations in a frequency range between 1 mHz up to about 0.1 Hz. This type of experiments allows determination of the dilational visco-elastic properties of liquid interfacial layers. Using a coaxial double capillary, experiments on sequential and simultaneous adsorption routes can be performed. This allows an analysis of the specific aspects of complex formation of e.g. proteins with other components or multilayer formation at the interface and determination of their dilational rheology. From a hydrodynamic point of view, the drop profile analysis methodology is limited to equilibrium drop/bubble profiles. Hence, when the size of a drop or bubble is changed too quickly their profiles are no longer Laplacian and the method would deliver wrong results. Therefore, the limits of PAT for a specific system need to be determined experimentally and validated by CFD simulations. Examples of CFD simulations have been presented in order to show how reliable the calculated interfacial tensions are.

This chapter provides an overview of dynamics and applications of pendant drops. The pendant drop tensiometry is widely used in measurement of interfacial tension and thus it is described in details including the historical development and the state-of-the-art. The sensitivity of the method is discussed and the recent advancements are presented. The stability and breakup of the pendant drop are also discussed in the general context of jet instability. The multiphysics effects including the electric field, viscoelasticity and surfactants are briefly reviewed focusing on their influence on pendant drop instability.

Multiple Drug Resistance (MDR) is acquired by bacteria, viruses, fungi, parasites and malignant tumours at interaction with antibiotics/medicines of old or new generation. Here is presented an up-date of MDR reported on bacteria, viruses, fungi, parasites and tumors, in this last field with emphasis on ophthalmology and neurology/neurosurgery. Multidrug resistant organisms exhibit in vitro resistance to one or more antimicrobial agents. One cause is the increasing use and misuse of antibiotics on humans and animals. Whereas particular bacteria are naturally resistant to some antibiotics, MDR occurs in other cases by accumulation of resistant plasmids and/or of genes, each gene determining resistance to a specific agent. The action of efflux pumps able to pump out more than one drug type is also a possible mechanism involved in MDR. In general, MDR is the most important “process” by which tumors acquire resistance to drugs during chemotherapy. Bacterial resistance to antibiotics used in ophthalmology has been reported since more than 10 years showing that several bacteria resistant to antibiotics were found in isolates from ocular infections. In neuroscience, development of new therapies to treat brain infections is more difficult. The most important cause of failure in developing new drugs for treating brain diseases is the existence and action of blood brain barrier (BBB). Brain tumors have usually poor prognosis and due to BBB, drug delivery to brain tumors is difficult. Some studies mention that BBB is involved in drug restriction to different brain neoplasias. The chapter concludes about the need to improve the arsenal conceived to overcome MDR acquired by different biological targets.

Since the interaction of a laser beam or otherwise called laser radiation with droplets in pendant position is a key process in the data reported in the book, the current chapter is dedicated to the description of the main properties of a laser beam of the kind used in presented experiments. Basically, laser emits electromagnetic radiation with particular properties described briefly in this chapter, such as: coherence (spatial and temporal), directivity coupled with mode structure, narrow (lower than 0.1 nm) spectral width, time structure of the emission (pulsed or continuous wave-cw), polarisation state (which is in most cases linear or closed to linear), Gaussian distribution of intensity in beam cross section, high beam energy, power and brightness.

The unresonant interaction between a single droplet and a single laser beam takes place when the beam is not absorbed by droplet’s material; it leads to specific effects induced in the droplet. The experiments reported here are made on droplets consisting in water solutions; 532 nm laser beam is used to produce unresonant interaction since water absorbs very little in green. The results on unresonant interaction, evidence several effects: (i) damped deformations and vibrations, (ii) expulsion of micro-jets of water that propagate with supersonic speeds and production of micro- and nano-droplets that are springs from mother droplet with different lower speeds, (iii) production of horizontal pillars of liquid and of cylindric channels within the droplet leading to appearance inside it of air bubbles. The water droplet interaction with a laser beam focused in its equatorial plane on droplet’s surface, produces a velocity gradient of water inside the droplet that may be experimentally evidenced. The velocity gradient increases with increasing beam energy and does not depend on droplet volume. The surface tension and liquid viscosity characteristics influence the effects produced by the laser beam on droplet and the propagation speeds of liquid material formations emitted out of them. Studies on unresonant processes have shown that if produced on a droplet that contains a laser dye, they may be accompanied by effects induced when the interaction is resonant, i.e. the laser radiation is absorbed by droplet material. Such a “combination” depends on laser beam energy, solvent, viscosity and liquid surface tension.

Resonant interaction of a laser beam with a single droplet takes place if the beam is absorbed in the droplet. Results are shown regarding the resonant interactions of pulsed laser beams with microliter droplets in pendant position which contain solutions of a laser dye-rhodamine 6G, phenothiazines (chlorpromazine, promazine), hydantoin derivative [(5Z)-5-(3-chlorobenzylidene)-2-thioxo-4-imidazolidinone)] and antibiotics (vancomycin), respectively, in water. A description of the electronic structure of rhodamine 6G and chlorpromazine using Gaussian09 and GaussView 5.0 software is made. Exposure of droplets to laser radiation leads to generation of new photoproducts that may have different properties if compared to parent compounds. Such modifications are evidenced by laser induced fluorescence and thin layer chromatography. In irradiated chlorpromazine water solution, out of hundreds of photoreaction products, 5 were identified: promazine (PZ), promazine sulfoxide (PZ-SO), 2-hydroxy promazine (PZ-OH), 2-hydroxy promazine sulfoxide (PZ-OH-SO), chlorpromazine sulfoxide (CPZ-SO). For rhodamine 6G solutions, the main results include the increasing intensity of LIF spectra in droplet with respect to bulk, even if the volume of the cuvette for bulk measurements is much higher than droplet’s volume. This behaviour may be explained by the confinement of light inside droplet, where total internal reflection at surface occurs, the droplet being associated with a spherical micro-optical resonator.

Precision measurements of surface tension and contact angle were performed on some medicines from the phenothiazine class (chlorpromazine, promethazine, and thioridazine) at different temperatures and concentrations in water, using the pending drop and the sessile drop methods. A specialised system DSA 100 (Krüss) was used for these measurements. Precautions were taken for an optimal acquisition of images (thermal isolation and heated windows of the chamber holding the studied droplets). Contact angles either static or dynamic were determined. Density measurements were also performed for the solutions. The same experiments were performed on these solutions after UV laser irradiation. The obtained values were discussed in comparison with the solvent behaviour and with the behaviour of other drugs (of alizarin dye class) as well. The average values of the advancing and receding angle, determined using a home developed program, are here presented for the first time.

The chapter presents a synthesis of results obtained on resonant interaction of laser radiation with medicines, in bulk, such as: cytostatics (methotrexate, 5-fluorouracil), phenothiazines (thioridazine, promethazine), quinazoline derivative (BG 1188) and hydantoin derivative (SZ-2). The effect of laser radiation on the studied samples is evaluated by analytical techniques out of which steady state absorption spectroscopy, laser induced fluorescence and FTIR spectroscopy, phosphorescence of generated photosensitised singlet oxygen and thin layer chromatography (TLC) used for qualitative photoproducts evidencing are approached. The molecular structural changes are suggested and the obtained photoproducts under laser exposure are analysed by spectroscopic means. This kind of data is related to a new method to combat multiple drug resistance acquired by microorganisms and tumours which develop new chemicals with bactericide or antitumour effects by exposing existing medicines that have low effects or are un-efficient, to laser beams.

This chapter presents results regarding production of emulsions and foams and their interaction with laser beams. Foams and emulsions are considered mainly collection of bubbles, or drops of different kind with particular properties, respectively. Here, emulsions of oily vitamin A and water in which various surfactants were introduced are described outlining the conditions in which they have a longer time stability. Laser induced fluorescence emitted by microdroplets that contain emulsions of rhodamine 6G in water and oily vitamin A is shown and the spectral distribution of the fluorescence radiation is described evidencing the enhancement of fluorescence emitted by droplets with respect to bulk. Lasing conditions in droplets are discussed and the role of reflections on foam drop inner structural components is outlined. Foams based on water solutions of vancomycin, produced by droplets interaction with laser beams are described in correlation with biomedical applications. Foams of polidocanol in water produced by Tessari method are described and their use in varicose vein treatments is introduced outlining the role of their exposure in tissues to infrared Nd:YAG laser beams in connection with the more rapid positive treatment effects. Polidocanol foam stability function of several surfactants such as tween 80, glycerin and xanthan gum is presented and discussed.

The development of new medicines and alternatives to existing antimicrobial agents represents a high priority in treating infections caused by multidrug resistant bacteria. This is reported mainly, as multiple resistance acquired by bacteria at treatment with antibiotics. The use of laser radiation to photo-generate new compounds from known medicines can be an effective method to obtain a better antimicrobial activity against infections with bacteria and fungi. The irradiation with a laser beam emitted at 266 nm of 2 mg/ml aqueous chlorpromazine and thioridazine solutions for periods of time varying from 1 to 240 min leads to photochemical changes in the molecular structure of the parental compounds and to generation of new photoproducts with enhanced antimicrobial, antifungal and antibiofilm activity. The susceptibility of broad panels of Gram-negative and Gram-positive bacteria and fungi in planktonic and biofilm state, to the unirradiated and irradiated CPZ and TZ were performed in order to highlight the possible use of these substances for the development of novel antimicrobial agents. The antimicrobial activity was evaluated by quantitative methods, i.e. minimum inhibitory concentration and minimum biofilm eradication concentration assays. Both CPZ and TZ irradiated solutions presented, as cocktails of medicines obtained after laser irradiation, enhanced antimicrobial, antifungal and antibiofilm activity when compared to the unirradiated samples.

Results are shown about the applications of medicines modified by exposure to optical radiation on pseudotumours indusced in rabbit eyes. These consist, first, in characterising modifications produced on medicines by exposing them to optical/laser radiation. Selected medicines are (i) cytostatics: methotrexate, 5-fluorouracil, (ii) benzopyridine derivatives: BG 204, BG 1120, (iii) phenothiazines: chlopromazine. They were used as solutions either in water or in natural saline. The anti-tumour properties of resulted solutions are measured after applying them on pseudotumours induced in rabbit eyes by Schmidt-Erfurth method. The use of methotrexate on pseudotumours has shown that eye conjunctive and neovascularisation disappear after 1-2 treatments which consists in exposure of eye injected with unirradiated methotrexate solution to cw Hg lamp radiation. Even if the use of methotrexate fastens the recovery, one should avoid direct exposure of eye to UV-Vis radiation. 5-fluorouracil was exposed to nitrogen pulsed laser beam which was sent to the eye already impregnated with cytostatics. Eye conjunctive and neovascularisation disappeared after 1-2 treatments, recommending 5-fluorouracil to cure such pseudotumours by combining its action with that of 337.1 nm laser. BG 1120 was used only exposed to cw Xe lamp incoherent radiation, without irradiating the eyes impregnated with solutions of it. BG 1120 modified by exposure to optical radiation leads to faster decrease of inflammations associated with pseudotumours and of neovascularisation of conjunctive tissue. For chlorpromazine, the most efficient in the recovery of a pseudotumour tissue is the sample irradiated 20 min, at 10 mg/ml concentration in water.

Photosensitive phenothiazine drugs subjected to UV laser radiation exhibit alteration of the initial parental compounds and provide newly generated photoproducts with possible bacteriostatic, bactericidal and antitumor effects. These new compounds with enhanced properties may also have outstanding wetting abilities when applied on fabrics of interest for applications, in order to develop new tools for drug delivery to patients, biological targets, or even surfaces of manmade equipment, constructions, or space ships. However, prior to biomedical applications, surface properties including contact angle measurements, require more thorough studies since such properties may control the delivery process of medicines to target tissues. This chapter shows the results obtained on the wettability of cotton, polyester and Parafilm M surfaces by laser irradiated phenothiazine aqueous solutions. In the present study, pendant chlorpromazine, promazine and promethazine droplets have been generated, then by simply bringing them into contact with the respective surfaces, detachment from a vertical capillary took place thus achieving the formation of sessile droplets. Results have evidenced the fact that some of the implemented drugs, containing the photoproducts obtained by prolonged exposure to laser radiation of the parent compounds, indicated better wetting abilities compared to their unexposed control, hence providing new promising perspectives.

The interaction of laser modified medicine solutions with hydrophilic and hydrophobic target surfaces has been investigated under the effect of simulated hypergravity conditions, employing the Large Diameter Centrifuge (LDC) facility, developed by the European Space Agency (ESA). Experiments have been performed within the HyperMed project under the aegis of the ESA “Spin Your Thesis!” 2015 programme. During centrifugation, real-time video files have been recorded regarding generation of ultrapure water, unexposed and laser exposed chlorpromazine aqueous pendant droplets, followed by their detachment due to the exerted high gravitational accelerations and finally by the formation of sessile droplets on target surfaces. In this way, information about the volume of the generated droplet, the degree of wetting and its time evolution at different hypergravity levels has been obtained. Phenothiazine solutions irradiated with UV laser radiation indicate reduced surface tension, thus presenting better wetting properties. Target surfaces impregnated with medicine solutions may constitute an unconventional tool and even vector in developing new drug delivery systems. Such a wetting process under high g-level conditions may be useful in space medicine applications. Microorganisms can survive, grow and even proliferate under the effect of increased gravity. Therefore, upon launching of a spacecraft, during a long-term mission in microgravity conditions, astronauts and spacecraft surfaces may require treatment and decontamination, respectively, against onboard infectious microbes. Since non-terrestrial gravity may alter drug properties, medicine droplets behaviour in interaction with target surfaces under hypergravity conditions is the aim of the present study.

Pendant droplets have optical properties that reproduce the characteristics of corresponding bulk materials. A particular case is the droplet hanging in air, made of a laser dye solution and optically side-pumped. Here are shown results about laser induced fluorescence (LIF) emitted by a single pendant droplet excited with 532 nm pumping pulsed laser beam when its content is solution of rhodamine 6G in water kept as such, or doped with TiO2. By changing the geometry of fluorescent medium from bulk to pendant droplet and its volume, significant LIF changes occur. The main difference is amplification of emitted fluorescence in pendant droplets, explained by the confinement of light via total internal reflection in droplet. A new technique is shown, developed to distinguish temporal changes of a droplet emission in air. It was observed that the presence in the droplet of rhodamine 6G solution in ultrapure water of TiO2 nanoparticles induces LIF spectra modifications depending on nanoparticles number density and laser beam pumping energy. A concentration of 1011 part/cm3 nanoparticles favours formation of two new emission bands shifted towards blue with respect to main band. The increase with one order of magnitude of TiO2 number density, produces the disappearance of these bands regardless the pumping energy. Data suggest that addition of TiO2 nanoparticles to rhodamine droplet solutions influences emission spectra which can be modulated by varying nanoparticles concentration and pumping beam energy.

In this chapter data about the laser induced fluorescence and Raman spectra obtained from microvolumetric (some microliters) droplets are introduced when they are exposed to laser radiation of suitable characteristics in terms of wavelength, power, and focusing conditions. A comparison is made with the same spectra obtained for the same solutions and using the same laser beams, except focus characteristics, from bulky volumes (some milliliters) showing that, basically, the information obtained from a single droplet after interaction with a single laser beam pulse is the same with that obtained from the bulky sample. In some cases, function of optimisation degree of the excitation geometry, the accuracy of spectral data collected from a microdroplet is higher than for bulk. The equivalence of the spectral data obtained from droplets and bulk is due to the fact that the interaction takes place in small volumes of the samples in both cases. In droplet, the remaining solution components that have not interacted with the pumping laser beam are very few, whereas in bulk their numbers are higher and so, the radiation emitted at the interaction volume is perturbed by them. This experimental evidence leads to the recommendation to use in many cases interaction of laser beams with a single droplet to obtain reliable spectral data, rather than bulky samples.