Gary B Braun
My research involves the design and synthesis of biomaterials and nanocomplexes that interface with cells. The materials are used in basic cancer research as remotely-activatable drug delivery vehicles, as probes in multiplexed imaging, flow cytometry and microfluidic diagnostics, and in high throughput screen assays to hunt for critical genes in tumor transport pathways.
Specialties: Bio-inorganic synthesis, design and surface chemistry. Fluorescence imaging, analytical chemistry, biochemistry, surface enhanced Raman spectroscopy (SERS), peptides and receptor targeting, plasmonics, pulsed lasers, mass-spec, nano-biological interfaces, NMR, AFM, TEM, FT-IR, DNA/RNA/protein surface engineering, endocytosis, tissue transport, long-circulating nanoparticles.
Specialties: Bio-inorganic synthesis, design and surface chemistry. Fluorescence imaging, analytical chemistry, biochemistry, surface enhanced Raman spectroscopy (SERS), peptides and receptor targeting, plasmonics, pulsed lasers, mass-spec, nano-biological interfaces, NMR, AFM, TEM, FT-IR, DNA/RNA/protein surface engineering, endocytosis, tissue transport, long-circulating nanoparticles.
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Papers by Gary B Braun
Surface enhanced Raman spectroscopy (SERS) is a powerful analytical technique that has been proposed as a substitute for fluorescence for biological imaging and detection but is not yet commercially utilized. The reason lies primarily in the lower intensity and poor reproducibility of most metal nanoparticle-based tags as compared to their fluorescence-based counterparts. Here, using a technique that scrupulously preserves the same number of dye molecules in both the SERS and fluorescence measurements, we show that SERS-based biotags (SBTs) with highly reproducible optical properties can be nanoengineered such that their brightness is at least equal to that of fluorescence-based tags.
We describe a near infrared (NIR) light-activated gene silencing method in undifferentiated human embryonic stem cell (hESC) using a plasmonic hollow gold nanoshell (HGN) as the siRNA carrier. Our modular biotin-streptavidin coupling strategy enables positively charged TAT-peptide to coat oligonucleotides-saturated nanoparticles as a stable colloid formation. TAT-peptide coated nanoparticles with dense siRNA loading show efficient penetration into a wide variety of hESC cell lines. The siRNA is freed from the nanoparticles and delivered to the cytosol by femtosecond pulses of NIR light with potentially exquisite spatial and temporal control. The effectiveness of this approach is shown by targeting GFP and Oct4 genes in undifferentiated hESC (H9). The accelerated expression of differentiation markers for all three germ layers resulting from Oct4 knockdown confirms that this method has no detectable adverse effects that limit the range of differentiation. This biocompatible and NIR laser-activated patterning method makes possible single cell resolution of siRNA delivery for diverse studies in stem cell biology, tissue engineering and regenerative medicine.
ABSTRACT: Reliable identification and collection of cells from bodily fluids is of growing interest for monitoring patient response to therapy, and for early detection of disease or its recurrence. We describe a detection platform that combines microfluidics with surface-enhanced Raman spectroscopy (SERS) for the identification of individual mammalian cells continuously flowing in a microfluidics channel. A mixture of cancerous and noncancerous prostate cells were incubated with SERS Biotags (SBTs) developed and synthesized by us, then injected into a flow-focused microfluidic channel which forces the cells into a single file. The spectrally rich SBTs are based on a silver nanoparticle dimer core labeled with a Raman-active small reporter molecule paired with an affinity-biomolecule, providing a unique barcode whose presence in a composite SERS spectrum can be deconvoluted. Individual cancer cells passing through the focused laser beam were correctly identified in among a proportionally larger number of other cells by their Raman signatures. We examine two deconvolution strategies:1 principal component analysis and classical least squares, to unmix the overall spectrum and determine the relative contributions of the two SBT barcodes: one indicating neuropilin-1 overexpression, the second a more universal tag that binds unspecifically to the cell's membrane. Highly reliable results were obtained for all of the cell mixture ratios tested, the lowest being 1 in 100 cells.
Abstract: We present a silica nanoparticle (SNP) functional- ized with polyphosphate (polyP) that accelerates the natural clotting process of the body. SNPs initiate the contact pathway of the blood-clotting system; short-chain polyP accelerates the common pathway by the rapid formation of thrombin, which enhances the overall blood-clotting system, both by accelerat- ing fibrin generation and by facilitating the regulatory anti- coagulation mechanisms essential for hemostasis. Analysis of the clotting properties of bare SNPs, bare polyP, and polyP- functionalized SNPs in plasma demonstrated that the attach- ment of polyP to SNPs to form polyP-SNPs creates a substan- tially enhanced synergistic effect that lowers clotting time and increases thrombin production at low concentrations. PolyP- SNP even retains its clotting function at ambient temperature. The polyP-SNP system has the potential to significantly improve trauma-treatment protocols and outcomes in hospital and prehospital settings.
While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.
Tumor-specific tissue-penetrating peptides deliver drugs into extravascular tumor tissue by increasing tumor vascular permeability through interaction with neuropilin (NRP). Here we report that a prototypic tumor-penetrating peptide iRGD (amino acid sequence: CRGDKGPDC) potently inhibits spontaneous metastasis in mice. The anti-metastatic effect was mediated by the NRP-binding RXXK peptide motif (CendR motif), and not by the integrin-binding RGD motif. iRGD inhibited migration of tumor cells and caused chemorepulsion in vitro in a CendR- and NRP-1-dependent manner. The peptide induced dramatic collapse of cellular processes and partial cell detachment, resulting in the repellent activity. These effects were prominently displayed when the cells were seeded on fibronectin, suggesting a role of CendR in functional regulation of integrins. The anti-metastatic activity of iRGD may provide a significant additional benefit when this peptide is used for drug delivery to tumors.
Neuropilins (NRPs) are trans-membrane receptors involved in axon guidance and vascular development. Many growth factors and other signalling molecules bind to NRPs through a carboxy (C)-terminal, basic sequence motif (C-end Rule or CendR motif). Peptides with this motif (CendR peptides) are taken up into cells by endocytosis. Tumour-homing CendR peptides penetrate through tumour tissue and have shown utility in enhancing drug delivery into tumours. Here we show, using RNAi screening and subsequent validation studies, that NRP1-mediated endocytosis of CendR peptides is distinct from known endocytic pathways. Ultrastructurally, CendR endocytosis resembles macropinocytosis, but is mechanistically different. We also show that nutrient-sensing networks such as mTOR signalling regulate CendR endocytosis and subsequent intercellular transport of CendR cargo, both of which are stimulated by nutrient depletion. As CendR is a bulk transport pathway, our results suggest a role for it in nutrient transport; CendR-enhanced drug delivery then makes use of this natural pathway.
Surface enhanced Raman spectroscopy (SERS) is a powerful analytical technique that has been proposed as a substitute for fluorescence for biological imaging and detection but is not yet commercially utilized. The reason lies primarily in the lower intensity and poor reproducibility of most metal nanoparticle-based tags as compared to their fluorescence-based counterparts. Here, using a technique that scrupulously preserves the same number of dye molecules in both the SERS and fluorescence measurements, we show that SERS-based biotags (SBTs) with highly reproducible optical properties can be nanoengineered such that their brightness is at least equal to that of fluorescence-based tags.
We describe a near infrared (NIR) light-activated gene silencing method in undifferentiated human embryonic stem cell (hESC) using a plasmonic hollow gold nanoshell (HGN) as the siRNA carrier. Our modular biotin-streptavidin coupling strategy enables positively charged TAT-peptide to coat oligonucleotides-saturated nanoparticles as a stable colloid formation. TAT-peptide coated nanoparticles with dense siRNA loading show efficient penetration into a wide variety of hESC cell lines. The siRNA is freed from the nanoparticles and delivered to the cytosol by femtosecond pulses of NIR light with potentially exquisite spatial and temporal control. The effectiveness of this approach is shown by targeting GFP and Oct4 genes in undifferentiated hESC (H9). The accelerated expression of differentiation markers for all three germ layers resulting from Oct4 knockdown confirms that this method has no detectable adverse effects that limit the range of differentiation. This biocompatible and NIR laser-activated patterning method makes possible single cell resolution of siRNA delivery for diverse studies in stem cell biology, tissue engineering and regenerative medicine.
ABSTRACT: Reliable identification and collection of cells from bodily fluids is of growing interest for monitoring patient response to therapy, and for early detection of disease or its recurrence. We describe a detection platform that combines microfluidics with surface-enhanced Raman spectroscopy (SERS) for the identification of individual mammalian cells continuously flowing in a microfluidics channel. A mixture of cancerous and noncancerous prostate cells were incubated with SERS Biotags (SBTs) developed and synthesized by us, then injected into a flow-focused microfluidic channel which forces the cells into a single file. The spectrally rich SBTs are based on a silver nanoparticle dimer core labeled with a Raman-active small reporter molecule paired with an affinity-biomolecule, providing a unique barcode whose presence in a composite SERS spectrum can be deconvoluted. Individual cancer cells passing through the focused laser beam were correctly identified in among a proportionally larger number of other cells by their Raman signatures. We examine two deconvolution strategies:1 principal component analysis and classical least squares, to unmix the overall spectrum and determine the relative contributions of the two SBT barcodes: one indicating neuropilin-1 overexpression, the second a more universal tag that binds unspecifically to the cell's membrane. Highly reliable results were obtained for all of the cell mixture ratios tested, the lowest being 1 in 100 cells.
Abstract: We present a silica nanoparticle (SNP) functional- ized with polyphosphate (polyP) that accelerates the natural clotting process of the body. SNPs initiate the contact pathway of the blood-clotting system; short-chain polyP accelerates the common pathway by the rapid formation of thrombin, which enhances the overall blood-clotting system, both by accelerat- ing fibrin generation and by facilitating the regulatory anti- coagulation mechanisms essential for hemostasis. Analysis of the clotting properties of bare SNPs, bare polyP, and polyP- functionalized SNPs in plasma demonstrated that the attach- ment of polyP to SNPs to form polyP-SNPs creates a substan- tially enhanced synergistic effect that lowers clotting time and increases thrombin production at low concentrations. PolyP- SNP even retains its clotting function at ambient temperature. The polyP-SNP system has the potential to significantly improve trauma-treatment protocols and outcomes in hospital and prehospital settings.
While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.
Tumor-specific tissue-penetrating peptides deliver drugs into extravascular tumor tissue by increasing tumor vascular permeability through interaction with neuropilin (NRP). Here we report that a prototypic tumor-penetrating peptide iRGD (amino acid sequence: CRGDKGPDC) potently inhibits spontaneous metastasis in mice. The anti-metastatic effect was mediated by the NRP-binding RXXK peptide motif (CendR motif), and not by the integrin-binding RGD motif. iRGD inhibited migration of tumor cells and caused chemorepulsion in vitro in a CendR- and NRP-1-dependent manner. The peptide induced dramatic collapse of cellular processes and partial cell detachment, resulting in the repellent activity. These effects were prominently displayed when the cells were seeded on fibronectin, suggesting a role of CendR in functional regulation of integrins. The anti-metastatic activity of iRGD may provide a significant additional benefit when this peptide is used for drug delivery to tumors.
Neuropilins (NRPs) are trans-membrane receptors involved in axon guidance and vascular development. Many growth factors and other signalling molecules bind to NRPs through a carboxy (C)-terminal, basic sequence motif (C-end Rule or CendR motif). Peptides with this motif (CendR peptides) are taken up into cells by endocytosis. Tumour-homing CendR peptides penetrate through tumour tissue and have shown utility in enhancing drug delivery into tumours. Here we show, using RNAi screening and subsequent validation studies, that NRP1-mediated endocytosis of CendR peptides is distinct from known endocytic pathways. Ultrastructurally, CendR endocytosis resembles macropinocytosis, but is mechanistically different. We also show that nutrient-sensing networks such as mTOR signalling regulate CendR endocytosis and subsequent intercellular transport of CendR cargo, both of which are stimulated by nutrient depletion. As CendR is a bulk transport pathway, our results suggest a role for it in nutrient transport; CendR-enhanced drug delivery then makes use of this natural pathway.