Resonance Raman Spectral Imaging of Intracellular Uptake of β-Carotene Loaded Poly(D, L -lactide- co -glycolide) Nanoparticles

DOI: 10.1002/cphc.201200577

Resonance Raman Spectral Imaging of Intracellular Uptake of b-Carotene Loaded Poly(D,L-lactide-co-glycolide) Nanoparticles Christian Matthus,[a] Stephanie Schubert,[b, c] Michael Schmitt,[e] Christoph Krafft,[a] Benjamin Dietzek,[a, c] Ulrich S. Schubert,[c, d] and Jrgen Popp*[a, c, e] The use of nanoparticles for drug delivery has been drawing considerable attention in pharmaceutical research. With increasing diversity and potential of various carrier systems, it is important to study the impact of nanocarriers on sub-cellular metabolic processes and organelles, since the delivery of a drug usually involves intra-cellular internalization. Herein, we employ Raman microscopy as a non-invasive method for cellular and sub-cellular imaging, to monitor the uptake and translocation patterns of particles based on poly(D,L-lactide-co-glycolide) over time. As the technique detects inherent signals

from the molecules of interest, it does not rely on external labels or dyes, which is an advantage over fluorescence labeling. For this purpose, the particles were loaded with b-carotene. The conjugated p-system of the molecule has a large Raman scattering cross-section and gives rise to resonance Raman effects, which can enhance the sensitivity by orders of magnitude. b-Carotene as a provitamin is not soluble in water and is thus usually of low bioavailability, which is enhanced by encapsulation into the nanoparticles.

1. Introduction Many therapeutic agents, in particular for gene and cancer therapy, target intra-cellular organelles, such as the nucleus, mitochondria, the Golgi or specific enzyme receptors. In order to pass the liposomal bilayer of the cell wall, a molecule needs to be to some extent lipophilic. For in vivo drug administration, a substance must also be hydrophilic in order to reach its site of action through the circulatory system. Most cytotoxicologically active compounds do not exhibit these amphiphilic properties and thus suffer from low bioavailability.[1] To circumvent the problem, the concept of active drug delivery has drawn increasing attention in pharmaceutical research.[2] Two possible approaches are to either chemically attach the active substance to a biomolecular vector or to encapsulate the substance into small particle-like structures, which can be administered as a suspension. Once taken up by the cell, the substance can be cleaved or dissociate into the cytosol. Whereas the vector molecules are usually readily recognized by cellular receptors, the carrier particles have to be designed in such a way that they can be taken up by the cells by various pathways and only subsequently release the active compound.[3–6] Numerous potential carrier systems have been developed and investigated for drug-delivery applications. Various biochemical compositions have been suggested. Among the early candidates were self-assembling structures based on lipids, which can form liposomes or micelles, in other words, artificial vesicles with, usually, a hydrophobic core and a hydrophilic shell. Their properties are easily controllable by their chemical composition.[1] Comparatively more stable materials are polymeric particles based on various biodegradable and non-biodegradable polymers or, for example, dendrimers.[7] The surface properties of these particles can also easily be manipulated by ChemPhysChem 2012, 13, 1 – 8

attaching for instance enzyme-specific antibodies, cell-penetrating peptides (CPPs), or simply by adding cationic lipids to the formulation.[8] The sizes of these nano-carriers are usually in the submicron range.[9–18] Uptake efficiency and intra-cellular fate are of particular interest for developing such carriers. The time frame for the particles being engulfed by the cell, as well as the release patterns and intra-cellular destination, depend strongly on the chemical composition. There are many pathways for small particles or vesicles to pass the cell wall. Depending mainly on size and surface properties, uptake can be driven by pinocytosis, endocytosis, phagocytosis, receptor-mediated or not receptor-mediated. The resulting intra-cellular vesicles can undergo various pathways, such as lysosomal degradation, secondary endocyto[a] Dr. C. Matthus, Dr. C. Krafft, Prof. B. Dietzek, Prof. J. Popp Institute of Photonic Technology (IPHT) Jena Albert-Einstein-Straße 9, 07745 Jena (Germany) [b] Dr. S. Schubert Laboratory of Pharmaceutical Technology Friedrich Schiller University Jena Otto-Schott-Straße 41, 07743 Jena (Germany) [c] Dr. S. Schubert, Prof. B. Dietzek, Prof. U. S. Schubert, Prof. J. Popp Jena Center for Soft Matter (JCSM) Friedrich Schiller University Jena Philosophenweg 7, 07743 Jena (Germany) [d] Prof. U. S. Schubert Laboratory of Organic and Macromolecular Chemistry (IOMC) Friedrich Schiller University Jena, Humboldtstraße 10 07743 Jena (Germany) [e] Prof. M. Schmitt, Prof. J. Popp Institute for Physical Chemistry and Abbe Center of Photonics Friedrich Schiller University Jena Helmholtzweg 4, 07743 Jena (Germany)

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J. Popp et al. sis or transportation back to the cell membrane for efflux. Endosomes can greatly vary in size, between about 20 nm to up to 500 nm.[19–21] The majority of studies visualizing the incorporation of nanoparticles into living cells employ fluorescence and electron microscopy.[22–24] Fluorescence microscopy is a very established tool in cell biology and is often the easiest approach since it requires a minimal amount of sample preparation. However, the fluorescence labelling often suffers from fast photo bleaching and low specificity, because of strong and spectrally broad backgrounds. Furthermore, the label itself can influence the biochemical behavior of the species of interest. Electron microscopy, on the other hand, requires invasive sample preparation and requires high electron density for optimum contrast. Recently, Raman microscopy has been extensively applied to testing the potential of the technique to study the chemical composition or compositional changes of cells or individual cell components.[25–30] With the development of the technique, it has become possible to characterize samples at the diffraction limit of the employed laser light.[31] Based on the associated spectral information, intra-cellular composition or metabolic activity may be obtained without any external labels or dyes. It also has been demonstrated that the intra-cellular uptake of fatty acids and nanoparticles can be monitored.[32–35] Particularly successful has been the use of stable isotopes. By exchanging hydrogen in a molecule with deuterium, the chemical properties of the labeled species remain unaltered, but the molecules of interest can be easily distinguished from their biochemical environment spectroscopically, since the CD stretching modes are shifted into the region between 2050 and 2200 cm1, where proteins generally have no Raman bands.[36, 37] Utilizing different carbon isotopes, Raman microscopy has been used to study the uptake of single-walled carbon nanotubes (SWNTs).[38] The Raman effect is a weak light scattering phenomenon, which excites molecular vibrations. Its sensitivity depends heavily on a high scattering cross-section. One possibility to enhance the intrinsically weak Raman effect is to utilize resonant electronic excitation of chromophoric units leading to an enhancement of the Raman scattering cross-section by about six orders of magnitude. By doing so the resonance Raman spectra are dominated by Raman modes which accompany the resonant electronic transitions, that is, modes of the electronic chromophore. We have recently used the ability of resonance Raman spectroscopy to measure low concentrated molecules within complex biological samples by investigating the carotenoid distribution in the chemical sphere around macroalgae and its axial distribution above the alga surface with diffraction limited spatial resolution.[39] Carotene compounds can easily be detected by resonance Raman spectroscopy even at low concentrations due to their high polarizability of the conjugated p-electron system and, consequently, high cross-section. b-Carotene is an unpolar tetraterpene and, thus, not water soluble. Its bioavailability is generally low and depends on consumption together with lipid nutrition.[40] Another possibility to enhance the Raman effect is surface-enhanced Raman

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scattering (SERS), which is based on the interaction of molecules with nanosized metal particles. SERS has also been successfully applied to various biomedical applications.[41] Among polymeric nanoparticles, particles based on poly(D,llactide-co-glycolide) (PLGA) are extensively investigated for drug-delivery applications.[42] PLGA polymers are biodegradable as well as biocompatible and have been approved for human use by the US Food and Drug Administration. Studies with various payloads have also shown promising delivery properties, such as controllable release kinetics and loading capacities.[43] Several intra-cellular uptake studies based on fluorescence microscopy as well as electron microscopy have been reported with, to some extent, conflicting results. It is generally assumed that uptake of PLGA particles is controlled by endo- and exocytosis, as well as lysosomal degradation.[44, 45] However, it has also been reported that PLGA particles are not readily taken up by cells, but deliver the drug loading by extracellular drug release or drug transfer, based merely on contact affinity between the particles on the cell walls.[46] Herein, we present results of uptake experiments based on Raman microspectroscopy, with enhanced sensitivity based on resonance Raman effects for b-carotene loaded PLGA nanoparticles. These studies further elucidate the potential of the technique for cellular/intracellular imaging and compare the results with findings of other microscopic techniques. b-Carotene is a member of the carotenoid family—a group of compounds that are generally of pharmaceutical interest because of their anti-oxidant activity. Due to their lipophilic nature, most carotenoids have low bioavailability. b-Carotene itself is mainly of interest for dermatological applications, although it has been suggested that it exhibits preventive effects against cancer, cardiovascular diseases, arteriosclerosis, macular degeneration and other age related diseases.[47–51] Other carotenoids as for example lutein and tretinoin are used for the treatment of acne.[52–55] For the studies presented here, b-carotene was chosen as an example derivative of the carotenoid family, which generally show interesting resonance Raman properties. With this contribution we would like to demonstrate the feasibility of utilizing resonance Raman effects for intracellular uptake experiments.

2. Results and Discussion b-Carotene loaded PLGA nanoparticles (and pure PLGA particles) were prepared via a single emulsion technique using sonication. The particle size and shape within the final suspension was characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM). The results correspond well, that is, the z-average diameter of b-carotene loaded particles with 620 nm and a polydispersity index of 0.430 (by DLS) fits well to the broadly distributed particles visualized by SEM (Figure 1) with particle sizes from 200 to 800 nm. The encapsulation efficiency is low with 0.63 mg per mg polymer particle, probably due to the ability of b-carotene to be mediated to certain extent into the aqueous media via the organic solvent, which is also evident in the orange supernatant during the washing procedure. Typical loading amounts for hydrophobic

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Resonance Raman Spectral Imaging

Figure 1. Scanning electron microscopy image of b-carotene-loaded PLGA nanoparticles. The scale bar represents 1 mm.

drugs in PLGA nanospheres are about 5 mg per mg polymer particle.[56, 57] Such loadings can be achieved after intense variation and optimization of the formulation conditions, which was not the scope of this research. Prior to the incubation experiments, resonance Raman spectra of b-carotene and the loaded nanoparticles were recorded at different excitation wavelengths to test for optimal Raman imaging conditions. The spectra acquired at 488, 514 and 785 nm were generally very similar (data not shown). The absorption spectrum of b-carotene in the visible range of light suggests maximum resonance effects between 450 and 550 nm. However, resonance Raman spectra have also been observed in the pre-resonance region and may be interpreted as a 2 1Ag optically forbidden transition. The absorption profile of the near IR region extends to about 800 nm. The nature of these transitions have not been resolved yet.[58–60] Excitation at shorter wavelengths introduced rapid photobleaching of b-carotene, leaving only Raman contributions from the PLGA. Consequently, the following results were obtained using a 785 nm excitation source. Figure 2 shows the spectra of pure b-carotene (A), b-carotene-loaded PLGA nanoparticles (B), and the PLGA nanoparticles (C). The spectrum of b-carotene is dominated by three main bands, which can be assigned to C=C stretches observed at 1510 and 1153 cm1 and a C-CH3 rocking

Figure 2. Resonance Raman and Raman spectra of b-carotene (A), b-carotene-loaded PLGA nanoparticles (B), and PLGA (C) excited at 785 nm.

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vibration at 1004 cm1.[61] These bands are still the most predominant in the spectrum of the carotene/PLGA particles, with smaller contributions from the spectrum of PLGA, as for instance the carbonyl stretching vibration of the ester bond at 1765 cm1 or the CH2 scissoring mode at 1448 cm1. The bands of b-carotene are observed at the same positions except for the band at 1510 cm1, which is shifted to 1520 cm1. The shift is possibly due to conformational changes in comparisson with the crystalline structure, caused by the embedding into the polymeric matrix. The cells were exposed to nanoparticles for a range of incubation times starting at 1 hour. Three hours were required to identify larger quantities of carotene/PLGA particles inside the cytoplasm reproducibly. Figure 3 illustrates the outcome of the VCA algorithm for a cell incubated for 3 h, showing the distri-

Figure 3. Spectra associated with intracellular abundances of b-carotene/ PLGA nanoparticles. Spectrum A-1 exhibits typical protein features, spectrum B-1 shows all bands associated with the b-carotene/PLGA nanoparticles, whereas C-1 shows the spectral characteristics of b-carotene and lipids in superposition, indicating an incorporation of the particles into intracellular vesicles. The abundance plots A-2, B-2 and C-2 reflect the intracellular distribution. The scale bar is 10 mm.

bution of the particles as well as the associated spectral information. Spectrum (A) reflects a typical protein spectrum, with spectral features that can mainly be assigned to vibrations of the protein backbone and aromatic amino acids. For example, the CH stretching vibrations are centred around 2940 cm1, the C=O double bound stretches at 1657 cm1, and the CH2 scissoring modes at 1450 cm1. The endmember spectrum is representative for the associated abundance plot in A-2. Bright areas correspond to a high protein density. The protein abundance profile reflects the cell body. The spectra shown in B1 and C-1 exhibit the very pronounced spectral features of bcarotene, still at the same positions at 1520 and 1153 cm1 as in Figure 2 B. Spectrum B-1 represents a spectrum of the unaltered carotene/PLGA particles. In spectrum C-1, the main spectral features of the b-carotene are still present and superim-

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J. Popp et al. posed with spectral features very typical for lipids, such as the C=O stretching of ester linkages at 1737 cm1, the C=C stretches of unsaturated aliphatic side chains at 1650 cm1 as well as other mainly CH and CH2 deformations at 1440, 1296, or 1260 cm1. The observed spectral characteristics are typical for intracellular vesicle like structures, such as endosomes, lysosomes or lipid droplet organelles.[62, 63] Here endosomal vesicles apparently encapsulate the particles taken up by the cell. The distribution of the b-carotene/PLGA particles inside the cytoplasm is shown in the associated images B-2 and C-2. In order to follow the intracellular uptake of the particles over time, the exposure times were extended to 6 and 9 h. The results for the time lapse experiments are shown in Figure 4. For better visualization, the images are pseudo colored. The abundances of spectral features associated with pro-

b-Carotene is insoluble in water, which makes a direct administration into the culture medium practically impossible. An injection of DMSO and THF solutions of b-carotene also led to immediate precipitation and formation of large aggregates. Therefore, a comparison of uptake efficiency between free bcarotene and b-carotene loaded into PLGA particles is not possible at this point. The addition of organic solvents to the culture medium is generally under criticism, because of toxicity and crystallization issues.[64]

3. Conclusions

The results clearly demonstrate the feasibility of Raman microscopy utilizing an enhanced scattering intensity due to resonance effects. The Raman spectra of the PLGA/b-carotene particles were overwhelmingly dominated by the spectral features of b-carotene, although the loading was only about 0.063 % (mg b-carotene per mg PLGA). Upon excitation at 785 nm, the scattering intensities of the bcarotene are at least an order of magnitude higher than the signal intensities of PLGA. Theoretically, the resonance Raman effect should give rise to higher intensities in the visible range, especially for blue and green excitations. Practically, resonance Raman effects can usually enhance the observed signal intensities by a factor of 104 and thus generate sensitivities comparable to fluorescence. However, very rapid photo bleaching was observed, which counteracts the increase in intensities. For the particle formulations Figure 4. Intracellular distribution of b-carotene/PLGA nanoparticles over time. Three cells per incubation time are reported here, an incubation shown as examples. Intracellular particle aggregation is evident after 3 h of incubation. The scale bar is 10 mm. period of 3 h was necessary to reach a level of saturation. Longer incubation did not result teins are plotted in light blue, whereas the abundances of the in an increase in cellular uptake. After that time frame, the particles are plotted in red. Similarly, as described for the PLGA/b-carotene nanoparticles are localized in vesicle-like above example, all cells show formation of droplet-like strucstructures throughout the cytoplasm. These vesicles can be retures after incubation with the nanoparticles. The inclusions solved at the diffraction limit of the employed laser light, but vary in size and reach a diameter of several micrometers. Three generally appear to be larger than usual endosomes. The assoexamples are shown for each time point. As apparent from the ciated spectral information shows different concentrations of images, the uptake does not increase after 3 h. The incorporatthe incorporated particles within these vessels. The varying ed amounts after 6 and 9 h incubation are similar. The associatamounts are reflected by different intensities of the main bed spectral information reflects an encapsulation for all incubacarotene bands superimposed with Raman bands mainly from tion times. There is no apparent dissociation of the b-carotene lipids. for the droplet-like vesicles. However, over time an increased Nanoparticles with biochemically unmodified surfaces can amount of fluorescence background was observed for the be taken up intracellularly with increasing size by pino-, endo-, spectra associated with the distribution of the particles, indior phagocytosis. After the formation of the respective vesicles, cating a slow degradation of the b-carotene. this process is often followed by secondary cytotic processes,

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Resonance Raman Spectral Imaging in which the vesicles can merge into larger ones. It is thus difficult to judge by the size of vesicle formations, which of the processes is dominant. Small endosomes are on the order of around 50 nm and cannot be resolved by conventional light microscopic techniques, such as fluorescence microscopy, because of the diffraction limit of light. However, fluorescence microscopic studies often indicate the presence of the labelled particles by a fluorescence background. It is often not possible to distinguish whether the actual particles or dissociation or leakage of the dye is the cause for this background. Employing Raman microscopy has the advantage that the detected signal is intrinsically associated with the molecules of interest. Based on fluorescence as well as electron microscopy, it has been shown that PLGA particles are taken up endocytotically and end up in lysosomes to a great extent.[45] Also exocytotic processes have been proven to become activated.[44] Recently, comparative studies of Nile red fluorescence labelling and CARS (coherent anti-Stokes Raman scattering) microscopy suggested that PLGA particles are not readily taken up by cells in vitro, but rather deliver their payload via fusion or contact at the cell walls.[46] Both may be true. However, the results presented here verify intra-cellular uptake of PLGA/b-carotene nanoparticles in larger quantities. Although early stages of endocytosis could not be resolved, the accumulation of particles in presumably late endosomes is evident, after 3 h of incubation. In general, Raman microscopy appears as a useful tool to study the uptake of compounds into cell cultured in vitro. It requires minimal sample preparation and provides reliable spectroscopic information. A major drawback to this technique in comparison with fluorescence microscopy is the relatively long measuring times. Although illumination times of 250 ms per spectrum may appear relatively short, an overall image still requires about 20 min.

Experimental Section Raman Data Acquisition Raman spectra were acquired using a WITec (Ulm, Germany) Model CRM Alpha-300Rplus confocal Raman microscope. Excitation (ca. 10 mW at the sample) was provided by a 785 nm diode laser (Toptica Photonics AG, Grfelfingen, Germany). The exciting laser radiation was coupled into a Zeiss microscope through a wavelengthspecific single-mode optical fiber. The incident laser beam was collimated via an achromatic lens and passed a holographic band pass filter before being focused onto the sample through the objective of the microscope. A Nikon Fluor water immersion objective (60  /1.00 NA, WD = 2.0 mm) was used in the studies reported here. The sample was located on a piezo-electrically driven microscope scanning stage with an x,y resolution of about 3 nm and a repeatability of  5 nm, and a z resolution of about 0.3 nm and  2 nm repeatability. The sample was scanned through the laser focus in a raster pattern at a constant stage speed of fractions of a micrometer per second. The continuous motion prevented sample degradation in the focal point of the laser beam. The spectra were collected with a 0.5 mm step size and an illumination time of 0.25 s, using a 300 mm grating. The spectral resolution was about 6 cm1 and the spectral window ranged from 300 to ChemPhysChem 0000, 00, 1 – 8

3200 cm1. At the applied conditions, an image required 20 min per cell.

Image Analysis and Data Processing Various algorithms for image analysis of hyperspectral datasets have been developed. For extracting spectral information, several factor methods such as principle component analysis (PCA) or vector component analysis (VCA) have shown high potential for the evaluation of Raman datasets.[62, 65] In principle, these algorithms search for a basis that describes the spectral variance optimally. Generally, the spectrum of a given image pixel is assumed to be a linear combination of the spectra of individual components: pij ¼

X

eik ckj

k

where pij is the ith band of the jth pixel, eik the ith band of the kth component spectrum and ckj is the mixing proportion for the jth pixel of the kth component. The mixing proportions are assumed to be percentages and the proportions should add up to one: X

ckj ¼ 1

k

This new basis then consists of a few vectors, or in our case Raman spectra, which can be used to reconstruct the dataset or image by plotting their individual abundances. The different algorithms vary mainly in what constraints are set for this change of basis. In PCA, for instance, the vectors have to be orthogonal. In VCA, the constraint is that the new vectors, usually referred to as endmembers, have to be all positive or real spectra. These endmembers normally represent the most dissimilar spectra within a dataset. For the calculation of the endmembers an N-FINDR algorithm was employed.[66, 67]

Particle Preparation b-Carotene-containing PLGA particles were prepared using a single emulsion oil/water (o/w) evaporation method similar to that in the literature.[68] Therefore, 2.5 mg b-carotene (purum  97 %, Sigma Aldrich) and 25 mg PLGA (MW = 7.000–17.000 g mol1, lactide:glycolide = 50:50, acid terminated, PLGA Resomer RG 502 H, Sigma Aldrich) were dissolved in 0.5 mL dichloromethane. An aqueous polyvinylalcohol solution (PVA, MW = 67.000 g mol1, 87–89 % hydrolyzed, Fluka) (1 mL, 3 wt % in dist. water) was added, and the mixture was sonicated for 4  1 s with each 1 s pause on an ice bath using a probe sonicator (Sonics VC505) with an amplitude of 30 %. The resulting emulsion was immediately poured into 10 mL 0.3 wt % PVA solution and stirred for 3 h at room temperature to allow the dichloromethane to evaporate completely. The particles were then isolated by ultracentrifugation (22 000 rpm, 15 min) and washed three times with distilled water by resuspension in a sonication bath following centrifugation. Shortly prior to cell studies, the particle suspensions were re-suspended in a sonication bath.

Particle Characterization The particle sizes were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Malvern, U.K.) operating with a laser beam at 633 nm and a scattering angle of 1738. The intensity distribution of the particle size was calculated from 15 runs for each 10 s at 25 8C. For further characterization, scanning electron microscopy images were obtained using a LEO-

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J. Popp et al. 1450 VP (Leo Elektronenmikroskopie GmbH, Oberkochen, Germany). The sputter coating device BAL-TEC SCD005 (Balzers, Lichtenstein; 60 mA, 80 s) was used. The system was operating at 8 kV. The encapsulation efficiency was determined via UV absorbance at 460 nm of dry particles (and b-carotene for calibration) dissolved in acetonitrile.

[22] [23] [24] [25] [26] [27]

Cell Culture and Incubation [28]

Murine NIH-3T3 cells (DSMZ, Braunschweig, Germany) were grown in 75 cm3 culture flasks (Fisher Scientific, Loughborough, Leicestershire, UK) with 15 mL of Dulbecco’s modified Eagle’s medium (DSMZ) and 10 % fetal bovine serum (DSMZ) at 37 8C and 5 % CO2. The cells were seeded onto and allowed to attach to polished CaF2 substrates (Crystal, Berlin, Germany), which were chosen to avoid background scattering in the Raman imaging experiments, observed from regular glass windows. The CaF2 substrates were removed from the culture medium after incubation with the nanoparticles, and the cells were fixed in a 5 % phosphate buffered formalin solution (Sigma–Aldrich) and washed in phosphate buffered saline. For the Raman and fluorescence studies, the CaF2 windows with the attached and fixed NIH-3T3 cells were submerged in a buffer solution during the measurements.

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Acknowledgements The Thringer Ministerium fr Bildung, Wissenschaft und Kultur (TMBWK, ProExzellenz-Programm NanoConSens), the “Europischer Fonds fr Regionale Entwicklung (EFRE)”, the “Thringer Ministerium fr Bildung, Wissenschaft und Kultur (TMBWK)” (Project B714-07037) and the Carl-Zeiss-Foundation (Strukturantrag JCSM) are highly acknowledged for financial support. Keywords: drug delivery · microscopy · nanoparticles · PLGA · Raman spectroscopy [1] V. Torchilin, Eur J Pharm Sci. 2000, 11, Suppl 2, S81 – 91 [2] R. Langer, Nature 1998, 392, 5 – 10. [3] C. Paulo, R. Pires das Neves, L. Ferreira, Nanotechnology 2011, 22, 494002. [4] M. Sui, W. Liu, Y. Shen, J. Controlled Release 2011, 155, 227 – 236. [5] S. Nimesh, N. Gupta, R. Chandra, J. Biomed. Nanotechnol. 2011, 7, 504 – 520. [6] S. Slomkowski, M. Gosecki, Curr. Pharm. Biotechnol. 2011, 12, 1823 – 1839. [7] R. Dhanikula, P. Hildgen, Bioconjugate Chem. 2006, 17, 29 – 41. [8] J. Wang, D. Mongayt, V. Torchilin, J. Drug Targeting 2005, 13, 73 – 80. [9] V. Torchilin, Annu. Rev. Biomed. Eng. 2006, 8, 343 – 375. [10] C. Plank, W. Zauner, E. Wagner, Adv. Drug Delivery Rev. 1998, 34, 21 – 35. [11] G. Kaul, M. Amiji, Pharm. Res. 2002, 19, 1061 – 1067. [12] G. Kaul, M. Amiji, Pharm. Res. 2005, 22, 951 – 961. [13] J. Panyam, V. Labhasetwar, Adv. Drug Delivery Rev. 2003, 55, 329 – 347. [14] J. S. Chawla, M. M. Amiji, Int. J. Pharm. 2002, 249, 127 – 138. [15] V. Torchilin, Nat. Rev. Drug Discovery 2005, 4, 145 – 160. [16] L. Serpe, M. Guido, R. Canaparo, E. Muntoni, R. Cavalli, P. Panzanelli, C. Della Pepal, A. Bargoni, A. Mauro, M. R. Gasco, M. Eandi, G. P. Zara, J. Nanosci. Nanotechnol. 2006, 6, 3062 – 3069. [17] N. Rao, V. Gopal, Biosci. Rep. 2006, 26, 301 – 324. [18] A. Bolhassani, Biochim. Biophys. Acta Rev. Cancer 2011, 1816, 232 – 246. [19] F. Zhao, Y. Zhao, Y. Liu, X. Chang, C. Chen, Y. Zhao, Small 2011, 7, 1322 – 1337. [20] M. D. Chavanpatil, A. Khdair, J. Panyam, J. Nanosci. Nanotechnol. 2006, 6, 2651 – 2663. [21] V. Torchilin, Handb. Exp. Pharmacol. 2010, 197, 3 – 53.

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Received: July 16, 2012 Revised: September 12, 2012 Published online on && &&, 2012

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ARTICLES C. Matthus, S. Schubert, M. Schmitt, C. Krafft, B. Dietzek, U. S. Schubert, J. Popp*

Non-invasive imaging: Cells were incubated with poly(D,L-lactide-co-glycolide)/b-carotene nanoparticles for different time periods. The figure shows a Raman image of a cell, with the distribution of the particles shown in red.

&& – && Resonance Raman Spectral Imaging of Intracellular Uptake of b-Carotene Loaded Poly(D,L-lactide-co-glycolide) Nanoparticles

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ÝÝ These are not the final page numbers!

ChemPhysChem 0000, 00, 1 – 8