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Nanotechnologies have opened up endless possibilities with applications at the molecular level from machines to medicines. The term 'nanomaterials' involves a wide range of materials including nanocrystalline materials, nanocomposites, nanoparticles, carbon nanotubes and nanosensors. These bulk materials incorporate nano-sized particles that are no longer than 100nm in any one direction. This particular characteristic of nanoparticles allows penetration of cells within living organisms. Living organisms are built of cells that are typically 10 um across, with subcellular components that are much smaller, usually in the sub-micron size domain. Beyond this, proteins have a typical size of just 5 nm, which is akin to the smallest manmade nanoparticles. Gaining a better knowledge base of biological processes on the nanoscale level, allows greater enhancements in the development of nanotechnology. The ability to deliver nanoparticles at the cellular level opens up great possibilities for many areas including drug delivery (e.g. protein drug delivery), genomic (DNA/siRNA) delivery and disease monitoring.
Nanoparticle drug delivery involves the active component (drug) being dissolved, entrapped, entrapped, encapsulated or attached to a polymer matrix. Depending upon the method of preparation, nanoparticles, nanospheres or nanocapsules can be obtained. Nanocapsules involve the encapsulation of the drug in a cavity surrounded by a unique polymer membrane, while nanospheres are matrix systems in which the drug is physically and uniformly dispersed. Recently, biodegradable polymeric nanoparticles, particularly those coated with hydrophilic polymer such as poly(ethylene glycol) (PEG) known as long-circulating particles, have been used as potential drug delivery devices because of their ability to circulate for a prolonged period of time and target a particular organ, as carriers of DNA in gene therapy, and their ability to deliver proteins, peptides and genes(Bhadra et al. 2002; Kommareddy et al. 2005; Lee and Kim 2005).
The ability to visualise intracellular processes by imaging has been a fast-paced technology concerning the imaging of live cells. There have been a number of approaches that have been proposed to achieve real-time, non-invasive analysis of chemical and physical properties with minimal distortion on the physiological status (i.e. perturbation). Major advances in this field have been achieved due to improvements in imaging processing technologies with high-performance computer systems as well as continuous development of new analyte-specific fluorescent molecular probes and optical fibre optodes(Lee and Kopelman 2009). Several drawbacks are associated with the use of molecular probes, which limits their use for intracellular imaging. The molecular probes must contain cell-permeable indicator dyes to allow for detection within the cell. The cytotoxicity of the indicator dyes may have a detrimental effect as well as variations in signal intensity caused by sequestration to specific organelles or by non-specific protein binding and other cell components. Perturbation of the indicator dyes due to typical penetration to the point of nucleus would be >1% of the cell volume limits the cellular viability. Furthermore, the dye is not usually 'ratiometric', due to the dye only having a single spectral fluorescence peak, which then requires technologically more demanding techniques, such as picosecond life time resolution or phase-sensitive detection(Lee and Kopelman 2009).
The limitations associated with fluorescent molecular probes and optical fibre optodes lead to the development of optical nanosensors. These devices are small enough to be inserted into living cells with minimal physical interference. The optical nanosensor is physically non-invasive owing to its small size. An optical nanosensor typically <100nm in diameter, and with a narrow size distribution, these sensors take up approximately 1ppb of a typical mammalian cell and cause minimum perturbation(Lee and Kopelman 2009).
Figure The design of an optical nanosensor(Lee et al. 2009).
Polyacrylamide-based optical nanosensors
The first optical nanosensor called PEBBLE (Probes Encapsulated By Biologically Localised Embedding) was introduced by Kopelman and co-workers(Kopelman et al. 1998). The polyacrylamide PEBBLEs are normally have dimensions less than 150 nm in any direction(Buck et al. 2004; Clark et al. 1999; Xu et al. 2002). The nanosensors are compromised of an inert, biofriendly polymer matrix in which chemical sensing elements such as fluorophores (bound to dextran) are entrapped. Multiple dyes can be entrapped within the matrix compared to single dyes being entrapped in fluorescent probes and optodes. This allows for multiple measurements to be made via fluorescence and ratiometric measurements to be observed. The key benefit of the polymer matrix is protection of the intracellular environment from any potential toxic effects of the sensing dye as well as the polymer matrix protecting the sensing dye from potential interactions such sequestration to specific organelles and non-specific protein binding(Xu et al. 2001). The polymer matrix is porous, allowing diffusion of analytes to interact with the entrapped fluorophores, resulting in a fluorescent response, which is captured by an optical system. The nanosensors can be surface coated with proteins or peptides via a linker molecule (usually a polymer, which also increases the plasma half-life) for targeting specific cells/intracellular sites.
Figure Polyacrylamide matrix synthesis of PEBBLEs(Di Benedetto et al. 2005)
Sol-gel based PEBBLE nanosensors
An additional matrix is also utilised for the use of optical nanosensors, namely silica sol-gel. The sol-gel matrix is used because of its superior properties over organic polymers. Sol-gel glass is a porous, high-purity, optically transparent and homogenous material(Uhlmann et al. 1997) . These characteristics make the sol-gel matrix ideal for use as a sensor matrix for quantitative spectrophotometric measurements. Silica-based sol-gel glasses efficiently embed organic molecules or probes for optical sensing within their matrices and are used extensively(Arduini et al. 2007). Sol-gel oxide-based are also used as stable host-matrices for the entrapment of specific reagents such as proteins and enzymes(Arduini et al. 2007).
These matrices possess many advantages with respect to alternative supports; the easiness of preparation allows preparation of matrices with specific physiochemical characteristics (i.e. pore size) by varying processing conditions and the amount or type of reactants used. Sol-gel matrices have low interaction with the analyte as well as optical transparency in the UV-vis range. Low processing temperatures enable easy incorporation of organic molecules and dyes, which are generally not stable at high temperatures. The matrices are also chemically inert and have high photochemical and thermal stability. Furthermore, the presence of OH groups on the surface favours the easy conjugation of a wide variety of molecules and functional groups. As reported by Arduini M et al(Arduini et al. 2007) dye-doped silica nanoparticles, nanostructures and sol-gel thin films, can act as optical sensors for Cu(II) ions in the micromolar range. Sol-gel PEBBLE nanosensors can vary greatly in particle size and are usually in the size range of 100-600 nm(Buck et al. 2004; Lin and Brown 1997; Xu et al. 2001).
The sol-gel matrix containing amino-functionalised groups is synthesised using a modified Stöber method(Balas et al. 2007; Stober et al. 1968) (Fig 3). The sol-gel matrix containing amino-functionalised groups are conjugated to the appropriate linker and Tat peptide.
Figure Synthesis of amino-functionalised silica nanoparticles(G. 2008)
Flourescent dyes - FITC-dextran and TAMRA-dextran
By using coimmobilisation techniques of two fluorescent dyes within a sensing matrix, ratiometric measurements can be undertaken. Ratiometric sensing is achieved by use of a reference fluorescent dye, 5-carboxy tetramethylrhodamine-dextran (TAMRA-dextran). Fig. 4 shows the structure of TAMRA.
Figure Structure of 5-carboxy tetramethylrhodamine(MERCK) Figure Structure of FITC(Scientific)
The fluorescence intensity of TAMRA-dextran is not affected by changing pH conditions, making it ideal as a reference dye. Dextran is conjugated to the dye, making it water soluble and has a high molecular weight, therefore aiding solubility within the polymer matrix when synthesising PEBBLEs and leaching out of the polymer matrix is kept to a minimum.
Flourescin isothiocyanate-dextran (FITC-dextran) responds to changes in pH and can be easily incorporated into the polymer matrix as it has been reported on many occasions on its use as an indicator dye(Coupland et al. 2009; Coupland et al. 2008; Webster et al. 2007). It is therefore an ideal candidate for use as an indicator dye.
Nanoparticle cellular uptake mechanisms
Optical nanosensors characteristics, give them the ability to overcome cellular barriers and penetrate cells at the intracellular level when conjugated to a cell penetrating molecule (Fig. 6). This is ideal for a drug delivery system whose target would be in an intracellular site for e.g. the nucleus for gene delivery.
Figure PEBBLE nanosensors attached to the cell penetrating peptide (TAT) targeting intracellular sites in a cell(Lee et al. 2009).
However it is not as straight forward in vivo, the physiochemical characteristics of the nanosensor and the nature of the target cells possess various internalization processes which take place which will affect the delivery of the drug. The two main internalization pathways are phagocytosis or the endocytic pathways (i.e. clathrin- and caveolae- mediated endocytosis)(Hillaireau and Couvreur 2009). Phagocytosis is an actin-based mechanism occurring primarily in professional phagocytes, such as macrophages, and closely associated with opsonisation.
Opsonisation involves attachment of proteins called opsonins to foreign nanoparticles (Fig 7A) recognised in the bloodstream rapidly after introduction into the human body. Opsonins are primarily immunoglobulins IgM and IgG as well as complement components C3, C4 and C6. The attachment of these proteins effectively enhances the uptake of the foreign nanoparticles. These opsonised nanoparticles then attach to macrophage cell by specific receptor-ligand interactions (Fig. 7B). Attachment to the receptor activates a signalling cascade mediated by the Rho-family GTPases, where actin assembly forms cell surface extensions which engulf the nanoparticle resulting in the formation of a phagosome (Fig 7C). The phagosome matures, fuses with lysosomes and become acidified due to the vacuolar proton pump ATPase located in the membrane leading to enzyme-rich phagolysosomes which are prone to nanoparticle degradation (Fig. 7D).
Figure Nanocarrier internalisation by opsonisation and phagocytosis(Hillaireau and Couvreur 2009).
Phagocytosis is occurs only in specialised cells where as other endocytic pathways occur in all virtually all cells. There are various mechanisms by which nanoparticles are taken up via other endocytic pathways. The clathrin-mediated endocytosis pathway is the main mechanism of internalisation for macromolecules and plasma membrane constituents. On binding of the nanoparticle/nanocarrier to the receptor, the assembly of clathrin triskeletons into a polygonal lattice which leads to release of the clathrin-coated vesicle into the cytosol after the membrane fuses. The uncoating of the polygonal lattice leads to the formation of an early endosome, which becomes acidified and is enzyme rich due to fusion with prelysosomal vesicles (Fig. 8B). The nanocarrier/nanoparticle is now prone to degradation (possible a drug delivery strategy but release from early endosome would be preferred).
Caveolae-mediated endocytosis is a more recent endocytic discovery which involves the formation of an caveolar vesicle (Fig 8C). Caveolae are flask-shaped membrane invaginations with dimensions being reported in the region of 60-100nm diameter range(Bareford and Swaan 2007; Conner and Schmid 2003; Mukherjee et al. 1997) which constitute 10-20% of the cell surface of predominately endothelial cells(Conner and Schmid 2003). Once the calveolar vesicle is formed in the cytosol it fuses with the caveosome which has an enzyme free environment which is advantageous for drug delivery by-passing any lysosomal degradation occurring.
Figure Various endocytic nanocarrier uptake mechanisms via: A) Macropinocytosis B) Clathrin-mediated endocytosis and C) Caveolae-mediated endocytosis(Hillaireau and Couvreur 2009).
Macropinocytosis is believed to be non-selective endocytic pathway which have dimensions in the region of >1um in diameter. This process occurs in many cells including macrophages. Actin-driven membrane protrusions form a micropinosome on fusion with the plasma membrane. These may eventually fuse with lysosomal compartments or simply recycle their content to the surface(Hillaireau and Couvreur 2009) (Fig 8A).
Another non-specific clathrin mediated endocytosis mechanism involves adsorptive pinocytosis typically referred as fluid-phase endocytosis. Uptake via this pathway does not require any interaction with membrane constituents instead resemble non-specific charges and hydrophobic interactions with the cell membrane. Clathrin-coated vesicles allow fluid entry which also internalise receptor ligands present within these pits as well as extracellular fluid and its content(Bareford and Swaan 2007).
Cell-penetrating peptides (CPP) - Tat mediated delivery
Small peptides (containing domains of less than 20 amino acids) that encompass cell membrane translocation properties have arisen over the last 10 years or so. Also called protein transduction domains and are known to have highly basic residues. These peptides have been utilised to deliver various conjugated cargoes of various molecular weights intracellularly to the nucleus or cytoplasm. A frequently employed CPP is one derived from the transcriptional activator protein encoded by human immunodeficiency virus type 1(Vives et al. 1997a), the Tat peptide (Tat protein). The Tat peptide has a highly basic minimal transduction domain of approximately nine amino acids (RKKRRQRRR)(Dennison et al. 2007). The Tat peptide has been used to internalise a range of cargoes including proteins, drugs and nanoparticles(Webster et al. 2007). The presence of a nuclear localisation signal (NLS) within its sequence allows efficient translocation of the peptide to the nucleus with accumulation present in the nucleoli(Vives et al. 1997b). However, although a significant amount research has been undertaken regarding the pathways proposed for Tat protein (including clathrin-dependant endocytosis, lipid-raft dependant macropinocytosis, and the use of non-endocytic pathways such as direct movement transmembrane(Dennison et al. 2007; Kaplan et al. 2005; Potocky et al. 2003; Richard et al. 2003)), it is still remains the subject of some controversy.
It has previously been reported that an amine-functionalised acrylamide monomer is incorporated into the nanosensor polymer matrix where a hetero-bifunctional linker such as sulfo-SMCC is used for attachment of the Tat peptide to the nanosensor surface.
Fig 9 Attachment of sulfo-SMCC to amine group on optical nanosensor and Tat peptide(Webster et al. 2005)
Polyethylene glycol (PEG) spacers allow synthesis of hetero-bifunctional linkers of varying lengths. PEG spacers improve water solubility of the reagent and conjugate and reduce the potential for aggregation of the conjugate (i.e. Tat peptide). There is increased flexibility within the crosslink resulting in a reduced immunogenic response to the spacer itself. The SM(PEG)12 is a heterobifunctional crosslinker with N-hydroxysuccinimide (NHS) ester and maleimide groups that allow covalent conjugation of amine- and sulfhydryl-containing molecules. Amine groups on the surface of the optical nanosensors covalently conjugate to the crosslinker as the NHS ester acts as a leaving group. The Tat peptide is attached to the crosslinker by a sulphur bond via the cysteine group on the Tat peptide.
Figure . SM(PEG)n reagent. For SM(PEG)12, n=12
The main aim of my investigation is to synthesise Tat conjugated nanosensors using two different hetero bifunctional linkers, SMCC and SM(PEG)12 using both polyacrylamide PEBBLEs and sol-gel PEBBLEs. Following synthesis, the optical nanosensors will be characterised for size and fluorescence (including pH calibration) using various techniques including fluorescence spectrometry, dynamic light scattering (DLS), CPS disc centrifugation, atomic force microscopy (AFM) and confocal microscopy. Two fluorescent dyes will be incorporated also into PEBBLE matrices to allow for pH calibration to be investigated via ratiometric analysis. The optical nanosensors will then be investigated for intracellular delivery to report their post-delivery environment and route of entry.
Acrylamide, N,N'-methylenebisacrylamide, ammonium peroxidisulfate (APS), 3-aminopropyl-trimethoxysilane (97%) (APTS), N,N,N',N'-tetramethylethylethylene-diamine (TEMED), Brij 30, dioctyl sulfosuccinate sodium salt (98%), Fluorescein isothiocyanate-dextran 10 000 MW (FITC-dextran), tetraethoxysilane 98% (TEOS), 5-tetramenthylrhodamine-dextran 10 000 MW (TAMRA-dextran) and Fluorecein isothiocyanate Isomer 1 were purchased from Sigma-Aldrich (St. Louis, USA). N-(3-aminopropyl)methylacrylamide hydrochloride was purchased from Polysciences INC., Warrington, PA. Ethanol (200 proof) and Hexane were purchased from Fischer Scientific (Loughborough, UK). Ammonia 50% was purchased from Alfa Aesar, Ward Hill, USA. Pureshield Argon was supplied by BOC. Sulfosuccincinimidyl-4-(N-msleidimidomethyl)-cyclo hexane-1-carboxy (sulfo-SMCC) was purchased from Calbiochem, USA. Tat peptide (CRRRQRRKKRG) was synthesised by the University of Nottingham Biomedical School peptide synthesis service with standard F-moc chemistry.
Polyacrylamide PEBBLE synthesis
The polyacrylamide nanosensors were produced by free-radical polymerisation of acrylamide in a reverse-phase microemulsion. The chemical sensing elements, FITC and TAMRA (dextran-bound), became physically entrapped within the polymer matrix during polymerisation.
Firstly hexane was deoxygenated with argon for approximately 1 hour. Meanwhile, the surfactants, 3.08 g of Brij 30 and 1.59 g of Dioctyl sulfosuccinate were measured out into a 100 ml round bottomed flask, ensuring an argon environment was maintained and then sealed and set up on a stirrer plate for 10 minutes. 42mls of deoxygenated hexane was added to the flask along with a 25 mm magnetic stirrer bar under argon to minimise oxygen contamination. Into a universal tube, 529.5 mg of acrylamide, 160 mg of N.N'-methylene bisacrylamide and 27.2 mg of N-(3-aminopropyl)methacrylamide hydrochloride were dissolved in 2 ml of deionised water with sonication. To this solution, aliquots of the fluorophores, 50 ul of FITC-dextran and 100 ul of TAMRA-dextran were added to 1850 ul of dissolved acrylamide solution into a separate vial. Under argon, this mixture was then added dropwise into the hexane/surfactant mixture. In a sealed argon environment, the reverse phase microemulsion was left to form for approximately 20 minutes. The polymerisation initiator solution, 30 ul 10% APS in deionised water and 15 ul TEMED was added to the above mixture and left for 2 hours (wrapped in foil). Hexane was removed by rotary evaporation until a viscous cloudy solution is obtained. 40 ml of ethanol was added to the viscous solution and transferred to two centrifuge tubes then spun on a centrifuge for 5 minutes at 6000 rpm for a repetition of six times ensuring all supernatant has been removed. Finally the nanosensors were resuspended in 10 ml ethanol and filtered using a Buchner funnel and anodised using a 200 nm filter membrane. Once collected, the PEBBLEs were now stored in the freezer at -18°C.
Sol-gel PEBBLE synthesis
The sol-gel PEBBLEs were synthesised using a modified Stöber method(Stober et al. 1968). Fig. 10 shows the formation of siloxane bonds incorporated in the nanosensors's core(Lin and Brown 1997). Synthesis was initiated by stirring a mixture of 5.8 ml of ethanol, 2.34 ml of ammonia 50 wt %, 1.56ml of deionised water, 300 ul of FITC-dextran and 500 ul of TAMRA-dextran in a round bottomed flask. The resultant solution was stirred until transparent, where 50 ul of (3-Aminopropyl)-trimethoxysilan 97% was added to the mixture then 450 ul of Tetraethoxysilan 98% dropwise to initiate hydrolysis of TEOS. The mixture was stirred further for 1 hour to allow the reactants to reach completion.
Figure 10 Synthesis of siloxane bonds incorporated in the core: 1. Hydrolysis of TEOS and formation of silanol groups, 2. Condensation of silanol groups to siloxane bonds(Lin and Brown 1997).
On completion of mixing, the solution was transferred in to a centrifuge tube and made up to 25 ml with ethanol, ensuring the round-bottomed flask was washed out with ethanol. Centrifugation was then carried out for 30 minutes at 6000 rpm. Fluorescence was undertaken of the supernatant to observe how much of the dyes were present (i.e. leached out/excess) and subsequently centrifugation was repeated until no more emissions of both FITC and TAMRA. The resultant solution was then filtered using a Buchner funnel and anodised using a 200 nm filter membrane. Once collected, the sol-gel PEBBLEs were stored in the freezer at -18°C.
Nanosensor surface functionalisation with Tat peptide
Surface attachment of the tat peptide was achieved through the use of a water soluble hetero-bifunctional linker, sulfo-SMCC or SM(PEG)12. 100 mg of the nanosensors (polyacrylamide PEBBLEs or sol-gel PEBBLEs) were dissolved in 5 ml of a 2 mM solution of sulfosuccinimidyl 4-N-maleimidomethyl cyclohexane-1-carboxylate (sulfo-SMCC) or succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol] ester (SM(PEG)12) respectively in PBS pH 7.4 and stirred for 1 hour at room temperature. The solution was then made up to 12 ml with ethanol and centrifuged for 2 minutes at 6000 rpm. The supernatant was removed and 4 ml PBS pH 7.4 was added and resuspended and 1 ml of 5 mg/ml tat peptide PBS pH 7.4 was added. The resultant solution was stirred for a further hour at room temperature and left overnight at 4°C. The nanosensors were then precipitated by centrifugation for 2 mins at 6000 rpm. Finally once re-suspended in minimal ethanol, they were filtered as described above and stored at -18 °C.
Fluorescence spectroscopic measurements were made of the incorporated fluorophores (FITC and TAMRA) using a spectrofluorometer for both types of PEBBLEs and for calibration of the PEBBLEs. The nanosensor samples were diluted prior to analysis. The fluorescence anlaysis was carried out using a Varian Cary Eclipse - Fluorescence Spectrophotometer. The slit values were set to 5 nm both excitation and emission. Emission spectra were collected using an excitation value of 490 nm for FITCand 545 nm for TAMRA.
Dynamic Light Scattering (DLS)
DLS was used to measure particle size distribution of the PEBBLEs. This was carried out using a Viscotek 802 DLS controlled by Omnisize software. Solutions were made up to a concentration of 0.5.mg/ml prior to analysis. The scattering intensity signal from the detector is passed to the correlator which compares scattering intensity at successive time internals to derive the rate at which the intensity is varying. The information is passed to the computer where the Nano software analyses the data and derives size information. Size distribution obtained was a plot of the relative intensity of light scattered by particles in various size classes therefore is known as the intensity size distribution. If more than one peak is present, conversion into mass or number used to look at relative importance of other peaks.
CPS Disc Centrifuge
Particles sediment in a fluid under a gravitational field according to Stokes' Law. Particles settle at a rate proportional to the square of their diameter. Particles of known viscosity can be used to determine particle size by measuring the time taken for the particles to sediment through the fluid. As the particles pass through the light source and detector on the edge of the disc, a turbidity measurement is used to convert the initial measurement into a weight distribution which then can be used to calculate a number distribution.
Size distributions were also determined using a CPS disc centrifuge particle size analyzer model DC240000 operating at 24000 rpm and with a sucrose gradient prepared on a gradient maker with 8% and 24% (w/v) sucrose solutions. The particle size analyzer was calibrated with a 377-nm PVC standard provided with the instrument. Size distributions were determined for PEBBLE nanosensors (100 ml) injected onto the gradient.
Figure 11 Front view of disc and cross section view of the disc(Europe)
pH calibration of PEBBLEs
A 0.1 M stock solution of citric acid was prepared by the addition 21.014 g of citric acid to 1 L of deionised water. A 0.2 M stock solution of sodium phosphate was prepared by adding 28.4 g of sodium phosphate to 1 L of deionised water. By utilising these stock solutions, the following pHs were made using the corresponding volumes of each stock solution.
Sodium phosphate (ml)
Citric acid (ml)
Figure 12 pH calibration solutions
12 mg of PEBBLEs were then added to 2 ml of each pH solution. Each sample was then run on a fluorescence spectrofluorometer, recording the emission and excitation values. A calibration curve was obtained by taking the emission value for FITC at each pH and dividing it by the emission value for TAMRA at the same pH. A graph of pH ratiometric intensity was then produced.
Atomic Force Microscopy (AFM)
AFM saw the interaction of polyacrylamide PEBBLEs and Tat peptide with the DPPC bilayers then visualised. Briefly, 1ml of lipid in chloroform was redissolved in 10mM Tris HCL buffer pH 7.4, which was then passed through ten freeze-thaw cycles using liquid nitrogen. Small volume extrusion apparatus containing a 100 nm polycarbonate filter was used to form vesicles (21 passes). A higher than ambient temperature was required for extrusion of DPPC (Tm 41 °C) than DOPC (Tm -20 °C), due the gel phase proving difficulties for extrusion.
Prior to AFM imaging, dilution of the suspension to 1 mg/ml was achieved by adding 10 mM Tris buffer and 100 mM MgCl2 , then 30 ul was added to freshly cleaved mica. DPPC were left at ambient temperature for not less than four hours to form bilayers. Using 10 mM MgCl2/ Tris buffer, the lipid bilayers were imaged in tapping mode using a Multimode AFM, with Nanoscope IIIa or V controller and liquid cell. AFM cantilevers with nominal spring constants of 0.58 N/m were ultilised with typical set point at 0.6-0.7 V, drive frequency 200-500 mV, and scan rate of 1 Hz. HPLC grade water filtered through a 0.2 um syringe filter was utilised for during synthesis of all solutions.
Polyacrylamide PEBBLEs (1 mg/ml) conjugated to Tat peptide via SMCC were added to the bilayer through the liquid imaging tip. Flattening of images (first order, plane-fit) was achieved by the use of Nanoscope software, and section analysis was undertaken to obtain image cross-sections and bilayer height.
Results and discussion
Synthesis of the optical nanosensors produced nanoparticles with the mean size range of approximately 20-400 nm in particle diameter as can be seen by the DLS data obtained below (Fig. 13-16). The ideal size range for optical nanosensors regarding delivery into an intracellular environment is more towards the lower end of the scale in the region of <100 nm. The addition of Tat peptide conjugated to SMCC and SM(PEG)12 and to the surface of the optical nanosensors played a slight effect regarding particle size as expected. Least variation in mean diameter was seen with the attachment of Tat peptide to SMCC, this would be expected as this is the shortest possible hetero-bifunctional linker as seen in Fig. 9.
For amino-functionalised sol-gel PEBBLEs with SMCC/Tat (Fig. 13), there was a slight increase in mean diameter from 348 nm to 408 nm, adding a total of 50 nm in diameter. This was expected as the addition of SMCC and Tat peptide to the surface of the PEBBLE would produce a larger overall diameter for the PEBBLE. For the amino-functionalised polyacrylamide PEBBLEs with SMCC/Tat (Fig. 14), a slight decrease was seen in mean diameter from 32.4 nm to 31.4 nm, a total decrease of 1 nm. This was unexpected as an increase in diameter would be expected with the conjugation of SMCC/Tat to the surface of the polyacrylamide PEBBLE. This can be explained possibly due to lack of or no attachment of SMCC to the surface of the PEBBLE.
Figure 13 DLS number distribution for Amino functionalised sol-gel PEBBLEs with Tat peptide attached via SMCC
Figure 14 DLS number distribution for Amino-functionalised polyacrylamide PEBBLEs with Tat peptide attached via SMCC
For amino-functionalised polyacrylamide PEBBLEs with SM(PEG)12/Tat, an increase was seen in mean particle diameter from 23.4 nm to 53.6 nm, a total increase of 30.2 nm in diameter (Fig 16). This was expected as SM(PEG)12 linker is much longer in length in comparison to SMCC causing an increase in overall diameter. However a larger increase in overall diameter was expected. The longer SM(PEG)12 linker also prevents aggregation occurring between the PEBBLEs leading to bigger particles. For amino-functionalised sol-gel PEBBLEs with SM(PEG)12/Tat, a decrease in mean particle diameter was observed from 116.8 nm to 42.2 nm in diameter (Fig 15). A total decrease in mean particle diameter of 74.6 nm. This was not expected due to the reasons stated above. This may be due to the formation of larger sized particles as can be seen by the intensity distribution. Formation of the larger sized nanoparticles may be due to aggregation occurring between the exposed amine groups on the surface of the PEBBLEs.
Figure 15 DLS number distribution for Amino functionalised sol-gel PEBBLEs with Tat peptide attached via SM(PEG)12
Figure 15a Intensity distribution for amino-functionalised sol-gel PEBBLEs with Tat attached via SM(PEG)12 . It can be seen particles of the following mean diameter: 42.4 nm, 298 nm, 1782 nm and 27092 nm.
Figure 16 DLS number distribution for Amino-functionalised polyacrylamide PEBBLEs with Tat peptide attached via SM(PEG)12
All the polyacrylamide PEBBLEs showed formation of larger aggregates on analysis of intensity distributions. Conjugation of Tat peptide via SM(PEG)12 to polyacrylamide PEBBLEs showed the most improved difference with the formation of fewer aggregates and a bimodal size distribution was obtained. Previous literature states the size of polyacrylamide nanoparticles can be varied by changing various synthesis conditions such as amount of surfactant, solvent, or monomer or the temperature of the reaction(Leong and Candau 1982). Using high concentrations of surfactants keeps the initial monomer micellar size very small which prevents formation of larger particles during polymerisation(Leong and Candau 1982).
It is important to note that the overall size (mean particle diameter) of the sol-gel PEBBLEs was greater than the polyacrylamide PEBBLEs. This was expected as literature states similar sizes in particle diameter were obtained in the region of 100-600 nm diameters for sol-gel nanoparticles(Arduini et al. 2007; Xu et al. 2001). Also for polyacrylamide PEBBLEs, sizes obtained were in the region of 20-60 nm in diameter which again comply with relevant literature(Buck et al. 2004; Clark et al. 1999; Xu et al. 2002).
CPS Disc centrifuge
Particle size distribution was measured for both the amino-functionalised sol-gel PEBBLEs conjugated to Tat peptide via SMCC and SM(PEG)12 (Fig. 17 ). For amino-functionalised sol-gel PEBBLEs it can be seen the average particle size is between 2.0-3.0 microns in diameter. This is larger than expected as literature states the size of sol-gel PEBBLEs should be in the region of 100-600 nm in particle diameter(Xu et al. 2001). This large particle size distribution may be due to aggregation between the amine groups on the surface of the nanosensors. The large size distribution was also reported by Xu, H et al(Xu et al. 2001) in their earlier experiments. To improve particle size distribution, the addition of PEG MW 5000 monomethyl ether was added during synthesis, which acts as a steric stabiliser in combination with a suction filtration system to aid separation of sol-gel particles reduced particle diameter distribution to 100-600 nm(Xu et al. 2001). There are also some sol-gel PEBBLEs with particle diameter in the region of 55nm, which is the region of interest thus little or no aggregation has occurred here and are of an ideal particle size for entry into intracellular environments. The attachment of Tat peptide via SMCC to the sol-gel PEBBLE produced smaller particles in comparison to the sol-gel PEBBLEs alone. This would be expected as the attachment of Tat peptide via SMCC to the surface of the sol-gel PEBBLE would produce less aggregation between the amine groups on the surface of the nanosensor. Attachment of tat peptide via SMCC would also produce a less dense nanosensor, which would therefore appear artificially smaller via disc centrifugation method as the larger, more dense particles will settle first (i.e. sol-gel PEBBLEs alone).
Figure 17 Particle size distribution for Amino functionalised sol-gel PEBBLEs with Tat peptide attached via SMCC
For amino-functionalised polyacrylamide PEBBLEs conjugated to Tat peptide via SM(PEG)12 again particle diameter was seen to be in the region of 2.0-3.0 microns for both type of PEBBLEs (i.e. with and without conjugation to tat peptide) (Fig18). This was not expected as for the reasons stated above. The conjugation of Tat peptide via SM(PEG)12 seem to produce particle with a slightly bigger particle diameter, again this was not expected as the attachment of SM(PEG)12 would be more likely to produce more dense particles due to the attachment of a longer hetero-bifunctional linker as a result of the extra 12 PEG additions within the linker. Therefore smaller particles should be observed through analysis with disc centrifuge if the SM(PEG)12 and Tat peptide was attached to the surface. Hence there was if any, very little attachment of SM(PEG)12 and Tat peptide to the surface of the sol-gel PEBBLE. The controversial attachment of SM(PEG)12 and Tat peptide also produced a wide particle size distribution, suggesting there was some attachement to the surface of the sol-gel PEBBLE where there is overlap with the sol-gel PEBBLE distribution.
Figure 18 Particle size distribution for Amino functionalised sol-gel PEBBLEs with Tat peptide attached via SM(PEG)12
Incorporation of FITC and TAMRA
Fluorescence spectrometry was carried out on both types of PEBBLEs employing either SMCC or SM(PEG)12 as a hetero-bifunctional linker. Fluorescence spectrometry saw excitation for FITC and TAMRA at the corresponding wavelengths of approximately 490 nm and 545 nm respectively for all synthesised optical nanosensors with slit values set to 5 nm for both excitation and emission. During initial synthesis of the sol-gel PEBBLEs, problems arose due to no or lack TAMRA was seen on the fluorescence spectra. This could be due to many reasons; including low concentrations of TAMRA-dextran were added during synthesis. Leaching of the dye from the sol-gel matrix could be a possibility as the dyes are unable to incorporate within the sol-gel matrix, which could be due to the incorrect conditions regarding the synthesis of the PEBBLEs.
Figure 19 Fluorescence Spectra for FITC and TAMRA - no incorporation of TAMRA was seen
Fluorescence spectra was obtained for all synthesised PEBBLEs and emission of FITC and TAMRA was seen in the region of 510 nm and 555 nm respectively as in Fig. 10.
Figure Fluorescence spectra for FITC and TAMRA incorporated into PEBBLEs
Incorporation of the dyes into the PEBBLEs could be seen visually during the synthesis of the PEBBLEs as the solution during synthesis would generally turn from clear to pink once the dyes have been added. At the filter stage prior to completion, the PEBBLEs would be of a pink shade once filtered and dried out, again this represents the incorporation of the dyes (FITC and TAMRA) into the PEBBLEs. A sample of the final product (PEBBLE) was then undergone analysis by the fluorescence spectrometer. Measurement of fluorescence spectra's of the pure dyes initially then of the PEBBLEs incorporating the dyes has been reported previously to characterise the nanoparticles(Arduini et al. 2007). A slight change in wavelength is normally observed towards the longer wavelength and this usually indicates the dyes have been incorporated into the polymer matrix under normal atmospheric conditions(Arduini et al. 2007).
pH calibration was carried out on the synthesised amino-functionalised polyacrylamide PEBBLES with SM(PEG)12. Ratiometric analysis uses the ratio of two fluorescent peaks instead a single absolute intensity peak. Ratiometric sensors are advantageous over single intensity peaks as factors such as excitation source fluctuations and sensor concentration will not affect the ratio between the fluorescence intensities of the indicator dye and reference dye(Clark et al. 1999; Xu et al. 2001). These slight fluctuations can be observed for the fluorescence spectra for FITC and TAMRA (Fig 21b).
The responses of the PEBBLES at different pHs are shown in Fig 21a, where FITC and TAMRA were excited by 490 nm and 545 nm respectively. The fluorescence emission peaks at 500 nm and 555 nm were assigned to FITC and TAMRA respectively. Therefore, the FITC/TAMRA fluorescence intensity ratio increases with pH and the pH can be reliably determined from the fluorescence intensity ratio in the range from pH 5.0 to pH 7.0, which is relevant for delivery of nanosensors intracellularly.
Figure 21a pH calibration of PEBBLE nanosensor
The fluorescence spectra shows that FITC has been incorporated into the PEBBLE nanosensor and that FITC is responsive to pH changes and TAMRA is insensitive to pH changes with only very slight fluctuations in intensity (Fig. 21b). These responsive changes in pH for FITC can be taken into account when investigating intracellular delivery, as there are many proposed routes of transport into the intracellular environment. For example, it has been reported that lysosomal pH is approximately 5.0(Mukherjee et al. 1997) and cytoplasmic pH is normally within the region of 7.0-7.2 pH(Hillaireau and Couvreur 2009). Taking the above pH's into account when investigating intracellular environments, the pH can be measured post-delivery and it can be seen which environment the nanosensor is in and possibly which route of uptake was taken.
Figure 21b Fluorescence spectra of FITC and TAMRA with different pHs
AFM was carried out using amino-functionalised polyacrylamide PEBBLEs utilising a SM(PEG)12 heterobifunctional linker conjugated to Tat peptide. DPPC bilayer is a saturated phosphatidylcholine(Leonenko et al. 2004). Initial imaging of the lipid bilayer provided an AFM image for DPPC prior to addition of polayacrylamide PEBBLEs as in Fig. The dark areas represent the mica surface whereas the coloured areas correspond to raised bilayer sections. DPPC undergoes a gel-fluid transition depending on operating conditions used for AFM(Leonenko et al. 2004). The transition temperature of DPPC is in the region of 41°C, therefore at room temperature in which the AFM analysis was carried out, DPPC is in the gel phase. Literature reports state that a height of 4.5-5.8 nm is usually associated with a DPPC bilayer(Leonenko et al. 2004) which is consistent with results obtained from the images below with a bilayer height of 5.167 nm (Fig 12). DPPC undergoes a gel-fluid transition depending on operating conditions used for AFM. The transition temperature of DPPC is in the region of 41°C, therefore at room temperature in which the AFM analysis was carried out, DPPC is in the gel phase.
Figure 22a DPPC section with bilayer height of 5.167 nm
Figure 22b Cross section of the surface topography presented in Fig. 11, showing bilayer thickness to be 5.167 nm
The polyacrylamide PEBBLEs conjugated to Tat peptide via SMCC were added to the DPPC bilayer after removal of excess buffer solution. The light pink areas on the surface of the DPPC bilayer are the polyacrylamide PEBBLEs conjugated to Tat peptide via SMCC. The height for the polyacrylamide PEBBLEs was shown to be 31.270 nm and a height of 4.126 nm was observed for the DPPC bilayer (Fig 23b). Previous literature suggest an increase in bilayer thickness was expected due as the conjugated Tat peptide inserts into the bilayer which leads to less space to hold the current conformation of order of lipids (Brooks et al. 2005; Dennison et al. 2007; Wang et al. 2008). As a result, the lipid molecules arrange themselves into a more ordered conformation to accommodate the nanoparticles conjugated to Tat peptide leading to an increase in bilayer thickness. However, a decrease in bilayer thickness was observed suggesting the nanoparticles did not adhere to the surface of the bilayer as well as expected, as can be seen by the AFM image (Fig 23a). This could be due alteration of the apparatus parameters, such as the set points and tapping hardness of the cantilever. Fluid phase formation of the bilayer may have taken place leading a decrease in bilayer height but unlikely as the environment was of ambient temperature. Attachment of the Tat peptide to the surface of the polyacrylamide PEBBLE via SMCC may also be an issue, where lack or no attachment occurred leading to low adherence levels to the bilayer.
During the formation of DPPC bilayers, the addition of MgCl2 is used to alter the zeta potential, making it more positive, allowing greater adhesion of liposomes to the surface of the mica(Egawa and Furusawa 1999). It can be seen visually that there are dense populations of the polyacrylamide PEBBLEs on the surface of the mica. The nanoparticles are basic in nature due the amine groups on the surface which haven't conjugated to Tat peptide via SMCC. Tat peptide is also basic in nature carrying multiple positive charges(Brooks et al. 2005). It has been previously reported that cationic nanoparticles have appeared to aggregate around membrane defects which seems to be in agreement with these findings(Dufrene and Lee 2000; Egawa and Furusawa 1999; Garcia-Manyes et al. 2006).
The particle size, 31.270 nm was consistent with the DLS data obtained for amino-functionalised polyacrylamide PEBBLEs conjugated to Tat peptide via SMCC, as the particle diameter was 31.4 nm mean diameter.
Figure 23a DPPC AFM image with polyacrylamide PEBBLEs conjugated to Tat peptide via SMCC
Figure 23b Cross section of the surface topography of Fig 13, showing DPPC bilayer thickness to be 4.126 nm and particle size to be 31.270 nm.
The first sol-gel and polyacrylamide PEBBLEs conjugated to Tat peptide via SM(PEG)12 have been produced here. DLS data obtained for sol-gel PEBBLEs and polyacrylamide PEBBLEs showed optical nanosensors were produced within the size range that is ideal for delivery into an intracellular environment, 100-600 nm and 20-60 nm respectively. However aggregates were produced for nearly all PEBBLEs and further synthesis of these optical nanosensors may be required altering synthesis conditions to produce more uniform particles with a narrow particle size distribution. Least aggregation was observed using the SM(PEG)12 hetero-bifunctional linker. Conjugation of Tat peptide to the PEBBLEs both increased and decreased the particle size depending on the hetero-bifunctional linker and PEBBLE used suggesting further investigation is required to clarify the effect the Tat peptide and hetero-bifunctional linker has on particle size. Incorporation of the dyes FITC and TAMRA was observed using fluorescence spectrometry. ph calibration saw the incorporation of the dyes; FITC and TAMRA and the responsiveness to changes in pH, this will be beneficial in terms of intracellular measurements and the route of uptake taken by the optical nanosensors. Further characterisation techniques are yet still to be completed such as transmission electron microscopy (TEM) to determine the size and shape of the PEBBLEs and confocal microscopy. Once characterisation is complete, the optical nanosensors will be investigated to measure intracellular environment(s) within a cell and the route of uptake taken by the nanosensors.
Synthesis of optical nanosensors utilising the SM(PEG)12 hetero-bifunctional linker has given an insight to the various possibilities available to us when synthesising nanosensors. By altering synthesis techniques, smaller and more uniform optical nanosensors can be produced and possibly opening up many advances in nanosensor techniques and drug design.