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Application of uv visible spectroscopy in pharmaceutical industry

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Farhad Ashraf

UV-visible spectroscopy can be used for clinical and pre-clinical applications in cancer diagnosis and analysis. It allows non-invasive measurement of biomarkers like tumor oxygenation, angiogenesis, redox state, and morphology. Spectroscopy measures how light interacts with tissue via absorption and fluorescence of chromophores and fluorophores. This optical signature contains physiologically meaningful information about cancer progression and treatment response that can provide insights not available from traditional pathology. Quantitative optical techniques have the potential to improve cancer management with personalized, molecularly-informed assessments.

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APPLICATION OF UV VISIBLE SPECTROSCOPY IN PHARMACEUTICAL INDUSTRY
SPECTROSCOPY • It is the branch of science which deals with the interaction of electromagnetic radiation with matter is called spectroscopy. • Or • It is the branch of science which deals with the study of interaction of matter with light.
UV SPECTROSCOPY • The interaction of electromagnetic radiation with matter when source is uv is called uv spectroscopy Spectrophotometer • Spectrophotometer are spectroscopic instrument that convert radiant intensities into electrical signal and measure the light passes through the sample • Spectrophotometer gives readings in transmittance (T) and absorbance (A)
ULTRAVIOLET-VISIBLE SPECTROPHOTOMETRY • UV-Visible spectrophotometry is one of the most frequently employed technique in pharmaceutical analysis. It involves measuring the amount of ultraviolet or visible radiation absorbed by a substance in solution. Instrument which measure the ratio, or function of ratio, of the intensity of two beams of light in the U.V-Visible region are called Ultraviolet-Visible spectrophotometers.
APPLICATIONS OF UV SPECTROPHOTOMETRY IN PHARMACEUTICALS • Qualitative analysis through spectrophotometric methods achieves fast and accurate results using only small sample quantities. This fast and effect instrumentation has become an essential tool in the pharmaceutical industry thanks to its adaptability and economic value. Qualitative analysis has proven highly useful in many major forms of organic compounds and helps to ensure patient health and safety.
QUANTITATIVE ANALYSIS OF PHARMACEUTICAL SUBSTANCES • Many drugs are in the form of raw material or in the form of formulation. They can be assayed by making a suitable solution of drug in a solvent and measuring the absorbance at specific wavelength. • Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at wavelength 284 nm.
UV-VISIBLE SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION OF ASSAY OF PARACETAMOL TABLET FORMULATION Paracetamol • Paracetamol or acetaminophen is a widely used over-the-counter analgesic (pain reliever) and antipyretic (fever reducer). It is commonly used for the relief of headaches and other minor aches and pains and is a major ingredient in numerous cold and flu remedies. In combination with opioid analgesics, Paracetamol can also be used in the management of more severe pain such as post-surgical pain and providing palliative care in advanced cancer patients. . However, acute overdose of Paracetamol can be potentially fatal and its toxicity is the leading cause of liver failure.
EXPERIMENT Materials • Paracetamol standard of was provided by Torque Pharmaceuticals (P) Ltd. (India). Paracetamol tablets containing 500 mg Paracetamol and the inactive ingredient used in drug matrix were obtained from market. Analytical grade methanol and water were obtained from Spectrochem Pvt. Ltd., Diluent preparation • Methanol and water (15:85, v/v) used as a diluent.
STANDARD PREPARATION • 10 mg drug was dissolved in 15 ml methanol and was shaken well. Then 85 ml water was added to it to adjust the volume up to 100 ml (100 ppm). From that 5 ml was taken and volume was adjusted up to 50 ml with diluents Test preparation • 20 tablets were weighed and powdered. Powdered tablet equivalent to 100 mg of paracetamol was weighed and taken into 100 ml volumetric flask then 15 ml of methanol was added and shaken well to dissolve it after that 85 ml of water was added to adjust the volume up to 100 ml. From that 1 ml of solution was withdrawn and taken in 100 ml volumetric flask. The volume was adjusted with diluent up to 100 ml. Instrumentation • UV-Visible double beam spectrophotometer with matched quartz cells (1 cm)
DEVELOPMENT AND OPTIMIZATION OF THE SPECTROPHOTOMETRIC METHOD • Proper wave length selection of the methods depends upon the nature of the sample and its solubility. To develop a rugged and suitable spectrophotometric method for the quantitative determination of paracetamol, the analytical condition were selected after testing the different parameters such as diluents, buffer, buffer concentration, and other chromatographic conditions. • Our preliminary trials were by using different compositions of diluents consisting of water with buffer and methanol. By using diluent consisted of methanol - water (50:50, v/v) best result was obtained and degassed in an ultrasonic bath (Enertech Electronics Private Limited). Below figures represent the spectrums of blank, standard and test preparation respectively.
SELECTION OF WAVELENGTH • Scan standard solution in UV spectrophotometer between 200 nm to 400 nm on spectrum mode, using diluents as a blank. Paracetamol shows λmax at 243. Conclusion • The present analytical method was validated and it meets to specific acceptance criteria. It is concluded that the analytical method was specific, precise, linear, accurate, robust and having stability indicating characteristics. The present analytical method can be used for its intended purpose.
SPECTROSCOPIC ANALYSIS ON THE BINDING INTERACTION OF BIOLOGICALLY ACTIVE PYRIMIDINE DERIVATIVE WITH BOVINE SERUM ALBUMIN Protein • Protein, one of the most important bioactive molecules, is related to alimentation, immunity and metabolism. The content of proteins in body fluid can be used as a vital index for the clinical diagnosis and health evaluation; therefore, the direct determination of protein is significant in life sciences, clinical medicine and chemical investigation. The interaction between bio-macromolecules and drugs has attracted great interest for several decades and many researches have been focused on two central questions about proteins: what are the determinant factors that influence the protein structures and functions, and how does a factor affect their biological activity
 Serum albumin (SA). • Serum albumin (SA), the main protein in the blood plasma acting as the transporter and disposition of many drugs, has been frequently used as a model protein for investigating protein folding and ligand binding mechanism.  Bovine serum albumin (BSA). • BSA is composed of three linearly arranged and structurally homologous sub-domains. It has two tryptophan residues that possess intrinsic domains (I–III) and each domain in turn is the product of two fluorescence: Trp-134, which is located on the surface of sub-domain IB, and Trp-212, located within the hydrophobic binding pocket of sub-domain IIA. The binding sites of BSA for endogenous and exogenous ligands may be in these domains and the principal regions of drugs binding sites of albumin are often located in hydrophobic cavities in sub-domains IIA and IIIA. So-called sites I and II are located in subdomain IIA and IIIA of albumin, respectively.
PYRIMIDINE • Pyrimidine moiety is one of the important classes of N- containing heterocycles widely used as key building blocks for pharmaceutical agents. It exhibits a wide spectrum of pharmacophore such as bactericidal, fungicidal, analgesic, anti-hypertensive and anti-tumor agents.
• Protein–drug interaction plays an important role in pharmacokinetics and pharmacodynamics. In a series of methods concerning the interaction of drugs and protein, fluorescence techniques are great aids in the study of interactions between drugs and serum albumin because of their high sensitivity, rapidity, and ease of implementation . The aim of the present investigation was to study the affinity of pyrimidine derivative (AHDMAPPC) for BSA using UV–visible and fluorescence spectroscopy to understand the carrier role of serum albumin for such compound in the blood under physiological conditions. Significantly, the determination and understanding of drug interacting with serum albumin are important for the therapy and design of drug . Knowledge of the interaction and binding of BSA may open new avenues for the design of the most suitable pyrimidine derivatives. All the experimental results clarify that AHDMAPPC can bind to BSA and be effectively transported and eliminated in body, which can be a useful guideline for further drug design.
MATERIALS AND METHODS • BSA and its molecular weight was assumed to be 66, 463 to calculate the molar concentrations. All BSA solutions (CBSA=2.0×10−5 M) were prepared in a pH 7.4 buffer solution and the stock solution was kept in the dark at 4 °C. Tris–HCl (0.1 M) buffer solution containing NaCl (0.1 M) was used to keep the pH of the solution at 7.4. A dilution of the BSA stock solution in Tris–HCl buffer solution was prepared immediately before use. The stock solution of AHDMAPPC (synthesized) was prepared in (5:95, v/v) ethanol water mixture. Dissolution of the compound was enhanced by sonication in an ultrasonic bath (Spectra Lab Model UCB-40). All chemicals were of analytical reagent grade and were used without further purification. Double distilled water was used throughout. In order to simulate human body fluid surroundings and to get the best sensitivity, Tris–HCl solution (pH 7.4) was chosen as the buffer solution in this work.
EQUIPMENT AND SPECTRAL MEASUREMENTS • The UV–visible absorption spectra were measured at room temperature on a Shimadzu UV–3600 UV–vis–NIR Spectrophotometer equipped with a 1.0 cm quartz cell. The wavelength range was from 250 to 450 nm. All pH values were measured by a digital pH-meter with magnetic stirrer (Equip- Tronics EQ-614A).
RESULTS AND DISCUSSIONS • BSA exhibited a strong fluorescence emission band at 347 nm. The fluorescence intensities of BSA reduced gradually with increasing AHDMAPPC concentrations, and a blue shift was also observed, which suggests that the fluorescence chromophore of serum albumin is placed in a more hydrophobic environment after the addition of AHDMAPPC.
UV-VISIBLE SPECTROSCOPY FOR CLINICAL AND PRE-CLINICAL APPLICATION IN CANCER • Methods of optical spectroscopy which provide quantitative, physically or physiologically meaningful measures of tissue properties are an attractive tool for the study, diagnosis, prognosis, and treatment of various cancers. Recent development of methodologies to convert measured reflectance and fluorescence spectra from tissue to cancer- relevant parameters such as vascular volume, oxygenation, extracellular matrix extent, metabolic redox states, and cellular proliferation have significantly advanced the field of tissue optical spectroscopy
INTRODUCTION • There is a great need to accurately quantify predictive biomarkers in vivo for the diagnosis, prognosis and treatment of cancers. Since current approaches in cancer management are generic across patients and involve empirical routines there is a growing emphasis toward developing individualized and personalized approaches which are based on detection of molecular, metabolic and physiological biomarkers. Traditional biomarkers include features such as the tumor grade, size and/or the number of local lymph nodes with metastasis
BIOMARKERS OF CANCER Vascular and metabolic factors • Oxygenation and hypoxia Oxygenation, particularly, the lack of it, is widely recognized as a crucial factor that influences the growth rate, metabolism, treatment resistance and metastatic behavior of cancer cells . Hypoxic microenvironments have routinely been identified in solid tumors of almost all tissues. Numerous studies have investigated the link between clinical outcomes and hypoxia using a variety of different methods to date . All of these studies have demonstrated that hypoxia is clearly related to clinical outcome, which motivates the importance of measuring it in vivo. • Currently, methods to measure tumor hypoxia can be divided into two classes, indirect and direct. The gold standard of direct tissue
ANGIOGENESIS AND BLOOD VOLUME • Irregular vasculature has previously been identified has a hallmark of cancer . Given that tumor cells have a constant need for new blood vessels to nourish their growth, solid tumors persistently sprout new segments of vessels to the existing vascular system leading to a highly irregular, leaky and chaotic network of blood vessels . The sprouting of new vessels is facilitated by over- expression of the vascular endothelial growth factor (VEGF), which is known to be upregulated under hypoxic conditions . This increased tumor vascularization eventually paves the way for a small, localized tumor to become an enlarged mass and subsequently metastasize to
REDUCTION-OXIDATION STATE OF THE CELL • Cellular respiration occurs via the electron transport chain in all aerobic cells and in the mitochondrial membranes of these cells, reactive oxygen species (ROS) are generated during oxidative phosphorylation. There are several complex cellular biochemical pathways that help cells protect themselves against low levels of ROS and free radicals by forming a network of redox buffers (which include the NAD(P)H/NAD(P)+ species). these mechanisms might be rendered dysfunctional under abnormally high levels of ROS leading to oxidative stress in the tumor microenvironment. onset of hypoxia within solid tumors causes the cells to prefer anaerobic glycolytic pathways over aerobic oxidative phosphorylation to meet their energy needs, which in turn influences both the amount of ROS produced, and the amount of NADH/NAD+ redox buffer available in these cells. There is currently no accepted clinical gold-standard to estimate the redox status of tumors, though detection in tumors has been achieved via the use of
MORPHOLOGICAL FACTORS • There are significant changes in cellular morphology and structure that are associated with the onset and progression of cancer. Pathologists routinely use microscopic differences observed in cellular and nuclear features including shape, size, crowding, chromatin organization and DNA structure in biopsied tissues to diagnose, prognosticate and stage disease.
OPTICAL SPECTROSCOPY • Methods of optical science and engineering have been developed for cancer detection and diagnosis and more recently to assess response to therapy in a variety of tissue sites for applications in both pre- clinical and clinical studies . The interaction of light with complex media such as biological tissues, is characterized by processes that depend on the physical nature of the light and the specific tissue morphology and composition . The incident light can be scattered (elastically or inelastically) multiple times due to microscopic differences in the index of refraction of cells and subcellular organelles within the tissues, and may be non-radiatively absorbed by chromophores present in the medium or by fluorophores, which release their excess energy by radiative decay, producing fluorescence. The remitted fluorescent light can, in turn, be multiply scattered or absorbed. Although complex, these optical responses can be measured by a variety of spectroscopic techniques and
• In optical spectroscopy, the wavelengths of illumination span the ultraviolet (UV) through the near-infrared (NIR) wavelengths. In steady-state reflectance spectroscopy, a broadband light source is used for illumination and a spectrum of the reflected light is collected , while in steady-state fluorescence spectroscopy a narrow spectral-band of incident light (obtained via filtering a broadband source or from a narrowband laser) is used to excite fluorophores and the emerging fluorescence spectrum at each excitation wavelength is detected

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Application of uv visible spectroscopy in pharmaceutical industry

  • 1. APPLICATION OF UV VISIBLE SPECTROSCOPY IN PHARMACEUTICAL INDUSTRY
  • 2. SPECTROSCOPY • It is the branch of science which deals with the interaction of electromagnetic radiation with matter is called spectroscopy. • Or • It is the branch of science which deals with the study of interaction of matter with light.
  • 3. UV SPECTROSCOPY • The interaction of electromagnetic radiation with matter when source is uv is called uv spectroscopy Spectrophotometer • Spectrophotometer are spectroscopic instrument that convert radiant intensities into electrical signal and measure the light passes through the sample • Spectrophotometer gives readings in transmittance (T) and absorbance (A)
  • 4. ULTRAVIOLET-VISIBLE SPECTROPHOTOMETRY • UV-Visible spectrophotometry is one of the most frequently employed technique in pharmaceutical analysis. It involves measuring the amount of ultraviolet or visible radiation absorbed by a substance in solution. Instrument which measure the ratio, or function of ratio, of the intensity of two beams of light in the U.V-Visible region are called Ultraviolet-Visible spectrophotometers.
  • 5. APPLICATIONS OF UV SPECTROPHOTOMETRY IN PHARMACEUTICALS • Qualitative analysis through spectrophotometric methods achieves fast and accurate results using only small sample quantities. This fast and effect instrumentation has become an essential tool in the pharmaceutical industry thanks to its adaptability and economic value. Qualitative analysis has proven highly useful in many major forms of organic compounds and helps to ensure patient health and safety.
  • 6. QUANTITATIVE ANALYSIS OF PHARMACEUTICAL SUBSTANCES • Many drugs are in the form of raw material or in the form of formulation. They can be assayed by making a suitable solution of drug in a solvent and measuring the absorbance at specific wavelength. • Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at wavelength 284 nm.
  • 7. UV-VISIBLE SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION OF ASSAY OF PARACETAMOL TABLET FORMULATION Paracetamol • Paracetamol or acetaminophen is a widely used over-the-counter analgesic (pain reliever) and antipyretic (fever reducer). It is commonly used for the relief of headaches and other minor aches and pains and is a major ingredient in numerous cold and flu remedies. In combination with opioid analgesics, Paracetamol can also be used in the management of more severe pain such as post-surgical pain and providing palliative care in advanced cancer patients. . However, acute overdose of Paracetamol can be potentially fatal and its toxicity is the leading cause of liver failure.
  • 8. EXPERIMENT Materials • Paracetamol standard of was provided by Torque Pharmaceuticals (P) Ltd. (India). Paracetamol tablets containing 500 mg Paracetamol and the inactive ingredient used in drug matrix were obtained from market. Analytical grade methanol and water were obtained from Spectrochem Pvt. Ltd., Diluent preparation • Methanol and water (15:85, v/v) used as a diluent.
  • 9. STANDARD PREPARATION • 10 mg drug was dissolved in 15 ml methanol and was shaken well. Then 85 ml water was added to it to adjust the volume up to 100 ml (100 ppm). From that 5 ml was taken and volume was adjusted up to 50 ml with diluents Test preparation • 20 tablets were weighed and powdered. Powdered tablet equivalent to 100 mg of paracetamol was weighed and taken into 100 ml volumetric flask then 15 ml of methanol was added and shaken well to dissolve it after that 85 ml of water was added to adjust the volume up to 100 ml. From that 1 ml of solution was withdrawn and taken in 100 ml volumetric flask. The volume was adjusted with diluent up to 100 ml. Instrumentation • UV-Visible double beam spectrophotometer with matched quartz cells (1 cm)
  • 10. DEVELOPMENT AND OPTIMIZATION OF THE SPECTROPHOTOMETRIC METHOD • Proper wave length selection of the methods depends upon the nature of the sample and its solubility. To develop a rugged and suitable spectrophotometric method for the quantitative determination of paracetamol, the analytical condition were selected after testing the different parameters such as diluents, buffer, buffer concentration, and other chromatographic conditions. • Our preliminary trials were by using different compositions of diluents consisting of water with buffer and methanol. By using diluent consisted of methanol - water (50:50, v/v) best result was obtained and degassed in an ultrasonic bath (Enertech Electronics Private Limited). Below figures represent the spectrums of blank, standard and test preparation respectively.
  • 11. SELECTION OF WAVELENGTH • Scan standard solution in UV spectrophotometer between 200 nm to 400 nm on spectrum mode, using diluents as a blank. Paracetamol shows λmax at 243. Conclusion • The present analytical method was validated and it meets to specific acceptance criteria. It is concluded that the analytical method was specific, precise, linear, accurate, robust and having stability indicating characteristics. The present analytical method can be used for its intended purpose.
  • 12. SPECTROSCOPIC ANALYSIS ON THE BINDING INTERACTION OF BIOLOGICALLY ACTIVE PYRIMIDINE DERIVATIVE WITH BOVINE SERUM ALBUMIN Protein • Protein, one of the most important bioactive molecules, is related to alimentation, immunity and metabolism. The content of proteins in body fluid can be used as a vital index for the clinical diagnosis and health evaluation; therefore, the direct determination of protein is significant in life sciences, clinical medicine and chemical investigation. The interaction between bio-macromolecules and drugs has attracted great interest for several decades and many researches have been focused on two central questions about proteins: what are the determinant factors that influence the protein structures and functions, and how does a factor affect their biological activity
  • 13.  Serum albumin (SA). • Serum albumin (SA), the main protein in the blood plasma acting as the transporter and disposition of many drugs, has been frequently used as a model protein for investigating protein folding and ligand binding mechanism.  Bovine serum albumin (BSA). • BSA is composed of three linearly arranged and structurally homologous sub-domains. It has two tryptophan residues that possess intrinsic domains (I–III) and each domain in turn is the product of two fluorescence: Trp-134, which is located on the surface of sub-domain IB, and Trp-212, located within the hydrophobic binding pocket of sub-domain IIA. The binding sites of BSA for endogenous and exogenous ligands may be in these domains and the principal regions of drugs binding sites of albumin are often located in hydrophobic cavities in sub-domains IIA and IIIA. So-called sites I and II are located in subdomain IIA and IIIA of albumin, respectively.
  • 14. PYRIMIDINE • Pyrimidine moiety is one of the important classes of N- containing heterocycles widely used as key building blocks for pharmaceutical agents. It exhibits a wide spectrum of pharmacophore such as bactericidal, fungicidal, analgesic, anti-hypertensive and anti-tumor agents.
  • 15. • Protein–drug interaction plays an important role in pharmacokinetics and pharmacodynamics. In a series of methods concerning the interaction of drugs and protein, fluorescence techniques are great aids in the study of interactions between drugs and serum albumin because of their high sensitivity, rapidity, and ease of implementation . The aim of the present investigation was to study the affinity of pyrimidine derivative (AHDMAPPC) for BSA using UV–visible and fluorescence spectroscopy to understand the carrier role of serum albumin for such compound in the blood under physiological conditions. Significantly, the determination and understanding of drug interacting with serum albumin are important for the therapy and design of drug . Knowledge of the interaction and binding of BSA may open new avenues for the design of the most suitable pyrimidine derivatives. All the experimental results clarify that AHDMAPPC can bind to BSA and be effectively transported and eliminated in body, which can be a useful guideline for further drug design.
  • 16. MATERIALS AND METHODS • BSA and its molecular weight was assumed to be 66, 463 to calculate the molar concentrations. All BSA solutions (CBSA=2.0×10−5 M) were prepared in a pH 7.4 buffer solution and the stock solution was kept in the dark at 4 °C. Tris–HCl (0.1 M) buffer solution containing NaCl (0.1 M) was used to keep the pH of the solution at 7.4. A dilution of the BSA stock solution in Tris–HCl buffer solution was prepared immediately before use. The stock solution of AHDMAPPC (synthesized) was prepared in (5:95, v/v) ethanol water mixture. Dissolution of the compound was enhanced by sonication in an ultrasonic bath (Spectra Lab Model UCB-40). All chemicals were of analytical reagent grade and were used without further purification. Double distilled water was used throughout. In order to simulate human body fluid surroundings and to get the best sensitivity, Tris–HCl solution (pH 7.4) was chosen as the buffer solution in this work.
  • 17. EQUIPMENT AND SPECTRAL MEASUREMENTS • The UV–visible absorption spectra were measured at room temperature on a Shimadzu UV–3600 UV–vis–NIR Spectrophotometer equipped with a 1.0 cm quartz cell. The wavelength range was from 250 to 450 nm. All pH values were measured by a digital pH-meter with magnetic stirrer (Equip- Tronics EQ-614A).
  • 18. RESULTS AND DISCUSSIONS • BSA exhibited a strong fluorescence emission band at 347 nm. The fluorescence intensities of BSA reduced gradually with increasing AHDMAPPC concentrations, and a blue shift was also observed, which suggests that the fluorescence chromophore of serum albumin is placed in a more hydrophobic environment after the addition of AHDMAPPC.
  • 19. UV-VISIBLE SPECTROSCOPY FOR CLINICAL AND PRE-CLINICAL APPLICATION IN CANCER • Methods of optical spectroscopy which provide quantitative, physically or physiologically meaningful measures of tissue properties are an attractive tool for the study, diagnosis, prognosis, and treatment of various cancers. Recent development of methodologies to convert measured reflectance and fluorescence spectra from tissue to cancer- relevant parameters such as vascular volume, oxygenation, extracellular matrix extent, metabolic redox states, and cellular proliferation have significantly advanced the field of tissue optical spectroscopy
  • 20. INTRODUCTION • There is a great need to accurately quantify predictive biomarkers in vivo for the diagnosis, prognosis and treatment of cancers. Since current approaches in cancer management are generic across patients and involve empirical routines there is a growing emphasis toward developing individualized and personalized approaches which are based on detection of molecular, metabolic and physiological biomarkers. Traditional biomarkers include features such as the tumor grade, size and/or the number of local lymph nodes with metastasis
  • 21. BIOMARKERS OF CANCER Vascular and metabolic factors • Oxygenation and hypoxia Oxygenation, particularly, the lack of it, is widely recognized as a crucial factor that influences the growth rate, metabolism, treatment resistance and metastatic behavior of cancer cells . Hypoxic microenvironments have routinely been identified in solid tumors of almost all tissues. Numerous studies have investigated the link between clinical outcomes and hypoxia using a variety of different methods to date . All of these studies have demonstrated that hypoxia is clearly related to clinical outcome, which motivates the importance of measuring it in vivo. • Currently, methods to measure tumor hypoxia can be divided into two classes, indirect and direct. The gold standard of direct tissue
  • 22. ANGIOGENESIS AND BLOOD VOLUME • Irregular vasculature has previously been identified has a hallmark of cancer . Given that tumor cells have a constant need for new blood vessels to nourish their growth, solid tumors persistently sprout new segments of vessels to the existing vascular system leading to a highly irregular, leaky and chaotic network of blood vessels . The sprouting of new vessels is facilitated by over- expression of the vascular endothelial growth factor (VEGF), which is known to be upregulated under hypoxic conditions . This increased tumor vascularization eventually paves the way for a small, localized tumor to become an enlarged mass and subsequently metastasize to
  • 23. REDUCTION-OXIDATION STATE OF THE CELL • Cellular respiration occurs via the electron transport chain in all aerobic cells and in the mitochondrial membranes of these cells, reactive oxygen species (ROS) are generated during oxidative phosphorylation. There are several complex cellular biochemical pathways that help cells protect themselves against low levels of ROS and free radicals by forming a network of redox buffers (which include the NAD(P)H/NAD(P)+ species). these mechanisms might be rendered dysfunctional under abnormally high levels of ROS leading to oxidative stress in the tumor microenvironment. onset of hypoxia within solid tumors causes the cells to prefer anaerobic glycolytic pathways over aerobic oxidative phosphorylation to meet their energy needs, which in turn influences both the amount of ROS produced, and the amount of NADH/NAD+ redox buffer available in these cells. There is currently no accepted clinical gold-standard to estimate the redox status of tumors, though detection in tumors has been achieved via the use of
  • 24. MORPHOLOGICAL FACTORS • There are significant changes in cellular morphology and structure that are associated with the onset and progression of cancer. Pathologists routinely use microscopic differences observed in cellular and nuclear features including shape, size, crowding, chromatin organization and DNA structure in biopsied tissues to diagnose, prognosticate and stage disease.
  • 25. OPTICAL SPECTROSCOPY • Methods of optical science and engineering have been developed for cancer detection and diagnosis and more recently to assess response to therapy in a variety of tissue sites for applications in both pre- clinical and clinical studies . The interaction of light with complex media such as biological tissues, is characterized by processes that depend on the physical nature of the light and the specific tissue morphology and composition . The incident light can be scattered (elastically or inelastically) multiple times due to microscopic differences in the index of refraction of cells and subcellular organelles within the tissues, and may be non-radiatively absorbed by chromophores present in the medium or by fluorophores, which release their excess energy by radiative decay, producing fluorescence. The remitted fluorescent light can, in turn, be multiply scattered or absorbed. Although complex, these optical responses can be measured by a variety of spectroscopic techniques and
  • 26. • In optical spectroscopy, the wavelengths of illumination span the ultraviolet (UV) through the near-infrared (NIR) wavelengths. In steady-state reflectance spectroscopy, a broadband light source is used for illumination and a spectrum of the reflected light is collected , while in steady-state fluorescence spectroscopy a narrow spectral-band of incident light (obtained via filtering a broadband source or from a narrowband laser) is used to excite fluorophores and the emerging fluorescence spectrum at each excitation wavelength is detected