Direct Raman imaging techniques for study of the subcellular distribution of a drug

Direct Raman imaging techniques are demonstrated to study the drug distribution in living cells. The advantage of Raman imaging is that no external markers are required, which simplifies the sample preparation and minimally disturbs the drug mechanism during imaging. The major challenge in Raman imaging is the weak Raman signal. In this study, we present a Raman image model to describe the degradation of Raman signals by imaging processes. Using this model, we demonstrate special-purpose image-processing algorithms to restore the Raman images. The processing techniques are then applied to visualize the anticancer agent paclitaxel in living MDA-435 breast cancer cells. Raman images were obtained from a cancer cell before, during, and after drug treatment. The paclitaxel distribution illustrated in these images is explained by means of the binding characteristics of the paclitaxel and its molecular target-the microtubules. This result demonstrates that direct Raman imaging is a promising tool to study the distribution of a drug in living cells.     

Paclitaxel-loaded microparticles for intratumoral administration via the TMT technique: preparation, characterization, and preliminary antitumoral evaluation

In our pursuit to develop suitable therapeutic particulate systems for intratumoral delivery by the targeted multi-therapy (TMT) technique, we describe the preparation of paclitaxel-loaded poly(D,L-lactic-co-glycolic) acid (PLGA) microparticles (MPs) (drug loading 35-38%, wt/wt; size 0.7-5 microm). Magnetite (15%, wt/wt) was also incorporated in some preparations for a future magnetic resonance imaging (MRI)-guided delivery. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) experiments showed that paclitaxel was not encapsulated in its initial crystalline form. The paclitaxel in vitro release pattern showed a biphasic tendency with a burst effect followed by a sustained release (28% released amount after 1 month), which was accompanied with MP erosion and degradation signs as confirmed by scanning electronic microscopy (SEM) micrographs. The paclitaxel-loaded MPs demonstrated a dose-dependent antitumor effect on human uterine cancer cells, with an IC(50) value relatively close to that of commercial Taxol. This paclitaxel delivery system represents a potent antiprofilerative and radiosensitizer agent for intratumoral administration via the TMT technique.     

Multi-wavelength raman imaging using a small-diameter image guide with a dimension-reduction imaging array

A commercially available fiber-optic Raman probe was modified for high-resolution spectral Raman imaging using a 350 microm diameter optical fiber image guide coupled to a dimension-reduction imaging array (DRIA). The DRIA comprised 672 optical fibers, arranged as a square array (21 x 32 fibers) on one end and a linear array (672 x 1 fibers) on the other. An imaging spectrograph was used with the DRIA to acquire multi-wavelength Raman images from -250 to 1800 cm(-1) at a spectral resolution of approximately 5 cm(-1). The utility of this technique for in situ and remote Raman imaging is demonstrated by monitoring the polymerization of a model polymer, dibromostyrene (DBS), while simultaneously measuring the Raman Stokes/ anti-Stokes ratio as a function of sample heating time, over a sample area of approximately 4 x 1.6 mm.     

Microfluidic Assembly of Monodisperse Multistage pH‐Responsive Polymer/Porous Silicon Composites for Precisely Controlled Multi‐Drug Delivery

We report an advanced drug delivery platform for combination chemotherapy by concurrently incorporating two different drugs into microcompoistes with ratiometric control over the loading degree. Atorvastatin and celecoxib were selected as model drugs due to their different physicochemical properties and synergetic effect on colorectal cancer prevention and inhibition. To be effective in colorectal cancer prevention and inhibition, the produced microcomposite contained hypromellose acetate succinate, which is insoluble in acidic conditions but highly dissolving at neutral or alkaline pH conditions. Taking advantage of the large pore volume of porous silicon (PSi), atorvastatin was firstly loaded into the PSi matrix, and then encapsulated into the pH‐responsive polymer microparticles containing celecoxib by microfluidics in order to obtain multi‐drug loaded polymer/PSi microcomposites. The prepared microcomposites showed monodisperse size distribution, multistage pH‐response, precise ratiometric controlled loading degree towards the simultaneously loaded drug molecules, and tailored release kinetics of the loaded cargos. This attractive microcomposite platform protects the payloads from being released at low pH‐values, and enhances their release at higher pH‐values, which can be further used for colon cancer prevention and treatment. Overall, the pH‐responsive polymer/PSi‐based microcomposite can be used as a universal platform for the delivery of different drug molecules for combination therapy.     

Semiconducting Polymer Nanocavities: Porogenic Synthesis,Tunable Host–Guest Interactions,and Enhanced Drug/siRNA Delivery

Nanocavities composed of lipids and block polymers have demonstrated great potential in biomedical applications such as sensors, nanoreactors, and delivery vectors. However, it remains a great challenge to produce nanocavities from fluorescent semiconducting polymers owing to their hydrophobic rigid polymer backbones. Here, we describe a facile, yet general strategy that combines photocrosslinking with nanophase separation to fabricate multicolor, water‐dispersible semiconducting polymer nanocavities (PNCs). A photocrosslinkable semiconducting polymer is blended with a porogen such as degradable macromolecule to form compact polymer dots (Pdots). After crosslinking the polymer and removing the porogen, this approach yields semiconducting polymer nanospheres with open cavities that are tunable in diameter. Both small molecules and macromolecules can be loaded in the nanocavities, where molecular size can be differentiated by the efficiency of the energy transfer from host polymer to guest molecules. An anticancer drug doxorubicin (Dox) is loaded into the nanocavities and the intracellular release is monitored in real time by the fluorescence signal. Finally, the efficient delivery of small interfering RNA (siRNA) to silence gene expression without affecting cell viability is demonstrated. The combined features of bright fluorescence, tunable cavity, and efficient drug/siRNA delivery makes these nanostructures promising for biomedical imaging and drug delivery.     

Method for quantitative determination of spatial polymer distribution in alginate beads using Raman spectroscopy

A new method based on Raman spectroscopy is presented for non-invasive, quantitative determination of the spatial polymer distribution in alginate beads of approximately 4 mm diameter. With the experimental setup, a two-dimensional image is created along a thin measuring line through the bead comprising one spatial and one spectral dimension. For quantitative analysis of the Raman spectra, the method of indirect hard modeling was applied to make use of the information contained in the entire recorded spectra. For quantification of the alginate signals from within the beads, a calibration curve acquired from sodium alginate solutions was used after it was shown that only negligible differences occur between signals from alginate solutions and alginate gels. The distribution of alginate over the bead gel matrix was acquired with high spatial (51 microm) and time (12 s) resolution. The inhomogeneous distribution obtained using the new measuring technique is qualitatively in excellent agreement with data from the literature. In contrast to known measuring techniques, correct quantitative information about the spatial polymer distribution within the matrix was derived. It gave an alginate mass fraction of approximately 0.045 g/g at the edges and 0.02 g/g in the center of the beads. Next to the determination of mere polymer concentrations, the excellent time resolution of the presented method will enable investigation of the dynamic process of gel formation and it will also serve as a basis for investigation of mass transfer of small diffusing molecules in alginate matrices.     

Lipid nanocapsules for intracellular drug delivery of anticancer drugs

As non-phagocytic eukaryotic cells can internalize particles < 1 microm in size, small size (25 to 110 nm) lipid nanocapsules (LNC) are proposed for the intracellular drug delivery of anticancer drugs to cancer cells. LNC of different diameters were loaded with etoposide or paclitaxel and subsequently tested for drug release kinetics and their efficiency to reduce cancer cell growth in cell culture. Relative high drug loads could be achieved and sustained drug release can be provided over a period of several days (etoposide) up to a few weeks (paclitaxel). While particle size exhibited only minor influences on the release kinetics, higher initial drug load led to a distinctly lower burst release. In a cancer cell culture model, etoposide or paclitaxel LNC showed a 4-fold or 40-fold higher efficiency, respectively than the drug solution while blank LNC were found to be less toxic than the pure drug at equivalent concentrations. The uptake and intracellular accumulation of LNC was confirmed by confocal laser scanning microscopy after fluorescence labeling of the nanocarriers. This nanoparticulate system is able to achieve efficient intracellular drug concentrations and seems to be therefore a promising therapeutic approach in cancer treatment.     

Resonance Raman imaging of macular pigment distributions in the human retina

We describe resonance Raman imaging (RRI) of macular pigment (MP) distributions in the living human eye. MP consists of the antioxidant carotenoid compounds lutein and zeaxanthin, is typically present in high concentrations in the healthy human macula relative to the peripheral retina, and is thought to protect this important central region from age-related macular degeneration. We demonstrate that RRI is capable of quantifying and imaging the spatially strongly varying MP distribution in the human retina. Using laser excitation of the MP molecules at 488nm, and sequential camera detection of light emitted back from the retina at the MP's strongest Raman peak position and at an off-peak position, RRI maps of MP are obtained at a resolution below 50microm within a fraction of a second per exposure. RRI imaging can be carried out with undilated pupils and provides a highly molecule-specific diagnostic imaging approach for MP distributions in human subjects.     

Quantitative image analysis of broadband CARS hyperspectral images of polymer blends

We demonstrate that broadband coherent anti-Stokes Raman scattering (CARS) microscopy can be very useful for fast acquisition of quantitative chemical images of multilayer polymer blends. This is challenging because the raw CARS signal results from the coherent interference of resonant Raman and nonresonant background and its intensity is not linearly proportional to the concentration of molecules of interest. Here we have developed a sequence of data-processing steps to retrieve background-free and noise-reduced Raman spectra over the whole frequency range including both the fingerprint and C-H regions. Using a classical least-squares approach, we are able to decompose a Raman hyperspectral image of a tertiary polymer blend into quantitative chemical images of individual components. We use this method to acquire 3-D sectioned quantitative chemical images of a multilayer polymer blend of polystyrene, styrene-ethylene/propylene copolymer, and polypropylene that have overlapping spectral peaks.     

Towards nanoporous polymer thin film-based drug delivery systems

A nanoporous polymer thin film has been developed as a potential platform for drug delivery. The film was fabricated by a light-induced polymerization process in which non-reactive solvent was first separated from photopolymer (dipentaerythritol penta-/hexa-acrylate as the monomer) and then removed from polymer via evaporation, yielding pores with diameters between 20 and 40 nm. Loading and release of Rhodamine B (drug model molecules) on both porous and non-porous thin films proved that nanopores enhanced the film's effectiveness in encapsulating and releasing the drug model molecules, which was attributed to the high surface-to-volume ratio of nanoporous film. Ultrasound-enhanced cumulative and pulsatile release revealed the advantages of ultrasound in controlled drug delivery.     

Characterization of polymeric micelles for pulmonary delivery of beclomethasone dipropionate

The potential of using poly-(ethylene oxide)-block-distearoyl phosphatidyl-ethanolamine (mPEG-DSPE) polymer to prepare BDP-loaded micelles with high entrapment efficiency and mass median aerodynamic diameter of less than 5 microm demonstrating sustained release properties was evaluated. The result showed that lyophilized BDP-loaded polymeric micelles with entrapment efficiency of more than 96% could be achieved. Entrapment efficiency was affected by both the drug to polymer molar ratio and the amount of drug used. Investigation using FTIR and DSC confirmed that there was no chemical or physical interaction and the drug was molecularly dispersed within the micelles. TEM images showed that the drug-loaded polymeric micelles were spherical in shape with multivesicular morphology. Further analysis by photon correlation spectroscopy indicated that the particle size of the BDP-loaded micelles was about 22 nm in size. In vitro drug release showed a promising sustained release profile over six days following the Higuchi model. The mass median aerodynamic diameter and fine particle fraction were suitable for pulmonary delivery. Moreover, the small amount of deposited drug in the induction port (throat deposition) suggested possible reduction in incidence of oropharyngeal candidiasis, a side effect normally associated with inhaled corticosteroids therapy. The high encapsulation efficiency, comparable inhalation properties, sustained release behavior together with biocompatibility nature of the polymer support the potential of BDP-loaded polymeric micelles as a versatile delivery system to be used in the treatment of asthma and chronic obstructive pulmonary disease.     

磁性水凝胶作为药物载体的应用研究进展

磁性水凝胶是一类同时具有磁性材料、高分子材料及水凝胶的性质特点的无机/有机复合材料。因具有优良的磁学性能及生物相容性,其作为新一代的药物载体可以实现磁响应、磁靶向及磁热疗等功能,在药物控制释放领域具有广阔的应用前景。对磁性水凝胶的制备方法及其在药物载体领域的研究情况进行了综述,详细介绍了磁性水凝胶作为药物载体的两种药物释放机理(ON/OFF模型及热敏释放原理),及其在磁靶向药物控释、磁热疗和磁共振成像方面的应用研究现状。     

Development of diclofenac sodium releasing bio-erodible polymeric nanomats

Application of nanofiber-based nanomats in medicine is attractive and thanks to the 3D nanostructure and the high surface to volume ratio they are excellent for local controlled drug delivery. The use of bioactive bioerodible polymers for developing drug delivery nanomats may allow for drug release and targeting control. Objective of the current study was to evaluate the suitability of bioerodible polymeric material based on n-butyl hemiester of [poly(maleic anhydride-alt-2-methoxyethyl vinyl ether)] (PAM14) for the preparation of nanomats for controlled administration of anti-inflammatory, diclofenac sodium (DS) drug. Samples were prepared using different polymer concentrations (5-10%) in either ethanol or acetic acid as solvent. Morphology was investigated by using scanning electron microscopy (SEM). Thermal analysis such as differential scanning calorimetry (DSC) was performed to detect effect on polymer arrangement. DS localization in electrospun nanomats was evaluated by using electron back scattering microanalysis, based on the detection of chlorine, and drug release kinetics was assessed using UV-Vis. Average fiber diameter resulted in the range of 100 nm to 1.0 microm and a homogeneous distribution of the loaded drug into the fibers was observed. The DS release was immediate and despite the preliminary nature of the performed electrospinning experiments, the achieved results appear promising for the future development of a novel system for the controlled and targeted administration of drug and active agent.     

Gadolinium Metallofullerene‐Based Activatable Contrast Agent for Tumor Signal Amplification and Monitoring of Drug Release

Activatable imaging probes are promising to achieve increased signal‐to‐noise ratio for accurate tumor diagnosis and treatment monitoring. Magnetic resonance imaging (MRI) is a noninvasive imaging technique with excellent anatomic spatial resolution and unlimited tissue penetration depth. However, most of the activatable MRI contrast agents suffer from metal ion‐associated potential long‐term toxicity, which may limit their bioapplications and clinical translation. Herein, an activatable MRI agent with efficient MRI performance and high safety is developed for drug (doxorubicin) loading and tumor signal amplification. The agent is based on pH‐responsive polymer and gadolinium metallofullerene (GMF). This GMF‐based contrast agent shows high relaxivity and low risk of gadolinium ion release. At physiological pH, both GMF and drug molecules are encapsulated into the hydrophobic core of nanoparticles formed by the pH‐responsive polymer and shielded from the aqueous environment, resulting in relatively low longitudinal relativity and slow drug release. However, in acidic tumor microenvironment, the hydrophobic‐to‐hydrophilic conversion of the pH‐responsive polymer leads to amplified MR signal and rapid drug release simultaneously. These results suggest that the prepared activatable MRI contrast agent holds great promise for tumor detection and monitoring of drug release.     

Broadband coherent anti-Stokes Raman spectroscopy characterization of polymer thin films

Broadband coherent anti-Stokes Raman spectroscopy (CARS) is demonstrated as an effective probe of polymer thin film materials. A simple modification to a 1 kHz broad bandwidth sum frequency generation (SFG) spectrometer permits acquisition of CARS spectra for polymer thin films less than 100 nm thick, a dimension relevant to organic electronic device applications. CARS spectra are compared to the conventional Raman spectra of polystyrene and the resonance-enhanced Raman spectra of poly(3-hexylthiophene). The CARS spectra obtained under these conditions consistently demonstrate enhanced signal-to-noise ratio compared to the spontaneous Raman scattering. The sensitivity of the CARS measurement is limited by the damage threshold of the samples. The dielectic properties of the substrate have a dramatic effect on the detected signal intensity. For ultrathin films, the strongest signals are obtained from fused silica surfaces. Similar to surface-enhanced Raman scattering (SERS), Au also gives a large signal, but contrary to SERS, no surface roughening is necessary.     

Crossed two-beam coherent anti-stokes Raman spectroscopy in dispersive media

Coherent anti-Stokes Raman spectroscopy (CARS) is a nonlinear optical wave mixing process that is used in gas-phase systems to determine the energy distribution of the probed species (usually N2) and, through a fitting procedure, the temperature giving rise to it. CARS signal strengths are maximized when the phase matching condition is met. Because gases are generally non-dispersive, this phase matching condition can be found geometrically as a function of the crossing angles between the CARS beams and their wavelengths. In addition, perfect phase matching in non-dispersive media occurs automatically for collinear beams. To improve spatial resolution, however, intersecting the laser beams is desirable. Being a third-order process, phase matching for CARS in gases typically requires three input laser beams. This paper discusses and demonstrates the issues of phase matching for CARS when the medium is dispersive, and the ability for CARS phase matching to occur with only two crossed laser beams (one pump and one probe). This two-beam X-CARS in dispersive media can be used as an alignment tool for gas-phase CARS and may be relevant as a simpler diagnostic in high-pressure environments. The paper also discusses the effects of non-ideal phase matching in dispersive and non-dispersive media.     

Surface enhanced coherent anti-stokes Raman scattering on nanostructured gold surfaces

Coherent anti-Stokes Raman spectroscopy (CARS) is a well-known tool in multiphoton imaging and nonlinear spectroscopy. In this work we combine CARS with plasmonic surface enhancement on reproducible nanostructured surfaces. We demonstrate strong correlation between plasmon resonances and surface-enhanced CARS (SECARS) intensities on our nanostructured surfaces and show that an enhancement of ～10(5) can be obtained over standard CARS. Furthermore, we find SECARS to be >10(3) times more sensitive than surface-enhanced Raman Spectroscopy (SERS). We also demonstrate SECARS imaging of molecular monolayers. Our work paves the way for reliable single molecule Raman spectroscopy and fast molecular imaging on plasmonic surfaces.     

CARS spectroscopy and optical interferometry of glow discharge plasma in nitrogen

The methods of spectroscopy of coherent anti-Stokes Raman scattering of light (CARS), optical interferometry, and emission spectroscopy are used to investigate the functions of distribution of nitrogen molecules over vibrational and rotational levels in the ground electron state. Also measured are the rate of gas heating and the distributions of gas and electron density and of translational temperature in the cross section of the discharge cuvette in plasma of dc glow discharge and contracted discharge. The obtained functions of distribution of nitrogen molecules over rotational and vibrational levels are well approximated by the Boltzmann and Treanor distributions, respectively. The radial dependence of the distribution of energy release in plasma of contracted discharge is suggested, and a solution is found of heat equation, which describes the distribution of translational temperature over the cuvette radius.     

Tumor‐Targeting Multifunctional Rattle‐Type Theranostic Nanoparticles for MRI/NIRF Bimodal Imaging and Delivery of Hydrophobic Drugs

The development of theranostic systems capable of diagnosis, therapy, and target specificity is considerably significant for accomplishing personalized medicine. Here, a multifunctional rattle‐type nanoparticle (MRTN) as an effective biological bimodal imaging and tumor‐targeting delivery system is fabricated, and an enhanced loading ability of hydrophobic anticancer drug (paclitaxel) is also realized. The rattle structure with hydrophobic Fe₃O₄ as the inner core and mesoporous silica as the shell is obtained by one‐step templates removal process, and the size of interstitial hollow space can be easily adjusted. The Fe₃O₄ core with hydrophobic poly(tert‐butyl acrylate) (PTBA) chains on the surface is not only used as a magnetic resonance imaging (MRI) agent, but contributes to improving hydrophobic drug loading amount. Transferrin (Tf) and a near‐infrared fluorescent dye (Cy 7) are successfully modified on the surface of the nanorattle to increase the ability of near‐infrared fluorescence (NIRF) imaging and tumor‐targeting specificity. In vivo studies show the selective accumulation of MRTN in tumor tissues by Tf‐receptor‐mediated endocytosis. More importantly, paclitaxel‐loaded MRTN shows sustained release character and higher cytotoxicity than the free paclitaxel. This theranostic nanoparticle as an effective MRI/NIRF bimodal imaging probe and drug delivery system shows great potential in cancer diagnosis and therapy.     

Imaging theory of nonlinear Raman confocal microscopy

A novel nonlinear Raman confocal microscopy utilizing Raman induced Kerr effect spectroscopy (RIKES) is presented in this paper. The imaging theory of RIKES confocal microscopy with Gaussian beam is derived. The imaging properties of RIKES confocal microscopy and the impact of different beam waist widths of Gaussian beam on the lateral and axial resolution have been analyzed in detail. It is proved that RIKES confocal microscopy has high sensitivity and high resolution, besides capability to characterize inherent structural features, such as vibration mode, vibration orientation, and optically induced molecular reorientation etc. Therefore, nonlinear Raman confocal microscopy that is based on RIKES has potential to provide a novel characteristic imaging method comparable to the existing imaging techniques based on other nonlinear optical processes, such as two-photon fluorescence, second harmonic generation (SHG) and coherent anti-Stoke Raman scattering (CARS).