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Dr. Rajendra Joshi

Address: Spemannstr. 41
72076 Tübingen
Room number: 4.B.05
Phone: +49 7071 601 705
Fax: +49 7071 601 702
E-Mail: Rajendra.Joshi

 

Picture of Joshi, Rajendra, Dr.

Rajendra Joshi

Position: Postdoctoral Fellow  Unit: Alumni Scheffler

My project mainly deals with the design and synthesis of novel intracellular targeted contrast agents that are specifically accumulating inside cells or tissue of interest. I am involved in the development of novel vectors based on e.g. peptides or lipids to facilitate the intracellular delivery of cargoes. Additionally, I am concerned with the synthesis of peptide nucleic acids which act as molecular sensors for the specific accumulation of the probes.

In another project silica nanoparticles are employed as platform for selective functionalization to couple targeting and imaging units in high amounts. For example MRI reporter units are coupled to enhance the sensitivity of contrast agents and cell penetrating peptides conjugation will facilitate intracellular delivery. This work is performed in collaboration with the group of Prof. H. A. Mayer at the University of Tuebingen within the project “Molecular in vivo imaging of cellular therapeutics – CeTheProbes” (BMBF FKZ 01EZ0813).

Project 1

Intracellular MR contrast agent composed of cholesterol and peptide nucleic acids

Introduction

Peptide nucleic acids (PNAs) are DNA mimics consisting of the four common bases of DNA on a pseudopeptide backbone. PNAs exhibit several attractive features like high biological stability and binding of RNA and DNA targets in a sequence specific manner. Therefore, they are widely used for antisense targeting of therapeutic drugs or probes for molecular imaging in cells. Unfortunately, the main hindrance to the effective use of PNAs has been their relatively poor uptake by cells.

Goals

The goal of the project is to develop novel antisense PNA conjugated intracellular MR contrast agents (CAs). Antisense PNA can hybridize uniquely to a complementary messenger RNA (mRNA) inside the cell providing specific accumulation of CAs in cells containing the targeted mRNA. Ultimately, imaging of mRNA transcription in cells and later in vivo by MRI would be possible by this means.

Methods

The synthesis of PNA conjugated to cholesterol and linkers was performed in a automated synthesizer using Fmoc continuous solid phase chemistry. All compounds were labeled with FITC to confirm cellular uptake. Cellular uptake was monitored in target expressing dsRed cells and their parent cells without target (CCL-11 mouse fibrosarcoma) by fluorescence spectroscopy and microscopy. MR imaging of both cell types was performed at 3T.

Initial Results

The synthesis of PNA, often laborious and lengthy, was simplified by developing and optimizing the scheme for an automated peptide synthesizer [1]. Various conjugates were synthesized altering the position of cholesterol, PNA and linkers in order to achieve better aqueous solubility as well as efficient internalization into cells [2]. Finally, the conjugate CA1 shown below (Fig 1) was synthesized and this conjugate was able to bind specifically to a synthetic 63mer target oligonucleotide in cell free gel shift assay. A subtoxic labeling concentration of 0.5 μM was sufficient to enhance significantly MR imaging contrast in both cell types (Fig 2) [3]. Fluorescence spectroscopy revealed a highly efficient and concentration dependent uptake of CA1 at very low labeling concentrations linearly increasing up to 3 μM. However at concentrations ≥ 3.0 μM precipitation was observed indicating a reduced solubility of these conjugates under physiological conditions. Fluorescence microscopy demonstrated that CA1 entered DsRed expressing cells and was mainly located in vesicles (green) around the nucleus (blue) (Fig 3) pointing toward a predominantly endosomal uptake mechanism. Because of the vesicular entrapment, it can be expected that there would be still a lack of interaction between CA1 and the target mRNA located in the cytosol as was previously reported for cell penetrating peptide-PNA conjugates [4, 5].

Initial Conclusion

The cholesterol conjugated CA1 was efficiently internalized into cells. A labeling concentration of 0.5 μM was sufficient to significantly enhance MR image contrast. However, modifications are still required to circumvent endosomal entrapment and to achieve a better cytosolic distribution of the CA and thus better specific accumulation in the target cells.

References

1.      Joshi, R., D. Jha, W. Su, J. Engelmann, Journal of Peptide Science, 17, 8-13 (2011).

2.      Joshi, R., R. Mishra, W. Su, J. Engelmann, Peptides 2008, Proceedings of 30th European Peptide Symposium, Helsinki, H. Lankinen (ed), pp. 550-551 (2009).

3.      Joshi, R., R. Mishra, R. Pohmann, J. Engelmann, Bioorganic & Medicinal Chemistry Letters, 20, 2238-2241 (2010).

4.      Su, W., R. Mishra, J. Pfeuffer, K.-H. Wiesmüller, K. Ugurbil, J. Engelmann, Contrast Media and Molecular Imaging, 2, 42-49 (2007).

5.      Mishra, R., W. Su, R. Pohmann, J. Pfeuffer, M. G. Sauer, K. Ugurbil, J. Engelmann, Bioconjugate Chemistry, 20, 1860-1868 (2009).

 

Fig 1. Chemical structure of CA1.

 

Fig 2. Cellular relaxation rate in DsRed and CCL-11 cells after labeling with CA1.

 

Fig 3. Cellular localization of CA1. Cells were incubated with 0.5 µM concentration in complete medium for 18 h. Green: FITC fluorescence of conjugate; blue: H 33342 (DNA stain); bar represents 20 µm.

 

Project 2

Silica particles with multiple functionalities for optical and MRI applications

Introduction

Magnetic resonance imaging (MRI) with its high temporal and spatial resolution is currently one of the most powerful noninvasive medical diagnostic tools. Contrast agents (CAs) are applied in many examinations to increase the specificity and sensitivity of MR imaging. Importantly, the development of such agents is gaining increased attention lately because these agents require optimization to generate maximum contrast.

Goals

Goal of the project is to differentially functionalize and modify the large surface area of nanoparticles (NPs) to achieve multifunctionality on a single particle. Because of these modifications large payload of cargo (e.g. gadolinium chelates) can be attached and anchoring moieties can be chosen to obtain different functions of interest (e.g. specific targeting, cell internalization).

Methods

Spherical, nonporous and monodisperse SiO2-NP (average Ø of 100 nm) functionalized with carboxylic acid groups [1] were reacted with Lys (Dde)-OH to make the exterior surface bifunctional. Selective couplings of GdDO3A-hexyl amine and fluorophores (FITC or Cy 5.5) alone or octaarginine containing FITC were performed (Fig 1). Cellular uptake was estimated by fluorescence microscopy in 3T3 mouse fibroblasts. MRI characterisation was done by suspending SiO2-NP in 1.5% agar (at 3T). In vivo studies were performed following the i.v. injection of NP3 into C57BL/6 mice (0.3 mg/200 μL/ 20 g body weight). Animals were sacrificed 10 min or 24 h later and optical imaging of ex vivo derived organs was performed.

Initial Results

The concentration of Gd3+ on the particles were determined by T1-measurements and the longitudinal relaxivity r1 was measured (Table 1). The low relaxivity in medium is caused most likely by the interaction of CO32- and PO43- with Gd(III) chelates. Dispersed particles showed a clear contrast enhancement in the agar layer (Fig 2). Voxelwise analysis confirmed a significant difference of signal intensity from each other and the control. Fluorescence microscopy demonstrated that these particles (NP1-2) entered the cells in a concentration dependent manner. Larger aggregates of NPs were located outside the cell membrane whereas smaller aggregates/single NPs could be detected inside the cells trapped in vesicles. The cellular internalization result was almost similar for both types of particles (with or without octaarginine).

In vivo studies of Cy 5.5 functionalized NPs (NP3) showed a strong fluorescence in the lung (red arrow in the Fig 3). Kidney and in particular the liver were also labeled after 10 min whereas only in the liver a sustained fluorescence could be detected after 24 h.

Initial Conclusion

A simple and straight forward method of modification and coupling of biomolecules on a bifunctional surface of spherical non porous silica support was developed. The applicability of such SiO2-NP as a bimodal probe for optical and MR imaging could be proven in initial biological studies.

Reference

Feldmann, V.,  J. Engelmann, S. Gottschalk, H. A. Mayer, Journal of Colloid and Interface Science, 366, 70-79 (2012)

Table 1. Longitudinal relaxivity r1 [mM-1s-1] of NP1-2 at 3T. Values represent mean ±SD.

 

Fluid

r1 (per mM Gd)

r1 (per mM nanoparticle)

NP1

water

5.6 ± 1.2

0.8 ± 0.2 x 105

medium

3.2 ± 1.3

0.5 ± 0.2 x 105

NP2

water

6.7 ± 0.9

1.1 ± 0.1 x 105

medium

3.9 ± 0.6

0.6 ± 0.1 x 105

 

 

Fig 1. Schematic structure of differentially modified SiO2-NP.

 

Fig 2. Axial T1-weighted MR images of NP1-2 in aqueous agar phantoms.

 

Fig 3. Fluorescence imaging of ex vivo organs after injection of NP3.

Since October 2007- Postdoctoral Research Scientist at the High-Field Magnetic Resonance Center, Max Planck Institute for Biological Cybernetics,   Tuebingen, (Germany). Working mainly on the development of novel intracellular contrast agents.

 

April 2004-April 2007- Ph D student at the University of Tuebingen (Germany).

 

Title-“Application of proteases for the total enzymatic synthesis of the Cholecystokinin octapeptide (CCK-8) using Benzoyl-Arginine as an enzymatically cleavable N-Terminal protecting group.”

 

October 2003-March 2004- Masters Thesis at the University of Tuebingen (Germany).

Worked on enzyme immobilization and peptide synthesis

 

 

1992-1998- Studies of Chemistry at Tribhuvan University, Kathmandu (Nepal).

 

 

Grants

2003-2007: DAAD Ph D fellowship

 

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Articles (5):

Joshi R Person, Feldmann V , Koestner W , Detje C , Gottschalk S Person, Mayer HA , Sauer MG and Engelmann J Person (January-2013) Multifunctional silica nanoparticles for optical and magnetic resonance imaging Biological Chemistry 394(1) 125–135.
Mishra A Person, Joshi R Person, Engelmann J Person and Logothetis NK Person (April-2012) Synthesis and in Vitro Evaluation of a Biotinylated Dextran-Derived Probe for Molecular Imaging ACS Chemical Neuroscience 3(4) 268–273.
Sweidan K Person, Engelmann J Person, Joshi R Person, Mubarak MS and El-Abadelah MM (October-2011) Synthesis of Some Cyclic Methylene 1,3-Diaza Barbiturates Derivatives Letters in Organic Chemistry 8(8) 603-605.
Joshi R Person, Jha D Person, Su W Person and Engelmann J Person (January-2011) Facile synthesis of peptide nucleic acids and peptide nucleic acid-peptide conjugates on an automated peptide synthesizer Journal of Peptide Science 17(1) 8-13.
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Joshi R Person, Mishra R Person, Pohmann R Person and Engelmann J Person (April-2010) MR contrast agent composed of cholesterol and peptide nucleic acids: Design, synthesis and cellular uptake Bioorganic and Medicinal Chemistry Letters 20(7) 2238-2241.

Conference papers (1):

Joshi R Person, Mishra R Person, Su W Person and Engelmann J Person (September-2008) CPP or cholesterol conjugation to antisense PNA for cellular delivery In: Peptides 2008: Chemistry of Peptides in Life Science Technology and Medicine, 30th European Peptide Symposium (30 EPS), FIPS, Helsinki, Finland, 550-551.
pdf

Posters (3):

Joshi R Person, Mishra R Person and Engelmann J Person (September-2009): Comparison of a lipid- vs. peptide-based delivery system for intracellular targeted MR imaging probes, 2009 World Molecular Imaging Congress (WMIC), Montréal, Canada.
Joshi R Person, Mishra R Person, Su W Person and Engelmann J Person (September-2008): CPP or Cholesterol Conjugated Antisense PNA for Cellular Delivery, 30th European Peptide Symposium (30 EPS), Helsinki, Finland, Journal of Peptide Science, 14(S1) 157.
pdf
Joshi R Person, Su W Person, Mishra R Person and Engelmann J Person (June-2008): Evaluation of Cholesterol Conjugation to Antisense PNA for Cellular Delivery, 2nd International Symposium "Cellular Delivery of Therapeutic Macromolecules 2008" (CDTM 2008), Cardiff, UK.
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Last updated: Friday, 17.01.2014