WO2010012816A1

WO2010012816A1 - Nanoparticles - external field effects

Info

Publication number: WO2010012816A1
Application number: PCT/EP2009/059908
Authority: WO
Inventors: Andre Skirtach; Helmuth MÖHWALD
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 2008-07-31
Filing date: 2009-07-30
Publication date: 2010-02-04

Abstract

The present invention relates to the interaction of external fields such as electromagnetic fields with absorbing nanoparticles, in particular, nanoparticle aggregates.

Description

Nanoparticles- External Field Effects

Description

The present invention relates to the interaction of external fields such as electromagnetic fields with absorbing nanoparticles, in particular, nanorods or nanoparticle aggregates. In particular, the invention relates to controlling membrane or film permeability, intracellular and in-vivo release, elasticity of surfaces and vesicles fusion caused by local temperature rise as well as to sensors and nanoparticle distribution. In all applications according to the invention nanoparticles - external field effects are used.

Nanoparticles (NPs) and nanomaterials find increasing application. They constitute the key building blocks of nanotechnology.

Nanoparticles can be used, for example, to functionalize membranes or films because of their unique optical and electronic properties. The permeability of membranes or films can be controlled by laser-nanoparticle interaction, which mechanism relies on surface plasmon resonance absorption and subsequent local temperature rise on nanoparticles upon exposure to laser light. Thus, nanoparticles, in particular, metallic nanoparticles, can be used to induce the release of encapsulated material by generating enough heat such that the capsules' shells are deformed. However, increase of permeability of membranes or films has been associated so far with the destruction of the membrane's or film's integrity.

Therefore, it was an object of the present invention to provide a method, wherein membrane or film permeability is controlled, while at the same time avoiding destructive heat generation.

According to the invention, this object is achieved by a method for reversibly controlling permeability of a membrane or film comprising nanoparticles, wherein an external field is applied to the membrane or film.

According to the invention it has been found that it is possible to reversibly control membrane or film permeability. In particular, it is possible to avoid destruction of the shells or films and, in particular, to avoid destructive heat generation. In contrast to earlier work it could now be shown that microcapsules can controllably release encapsulated material upon application of an external field and the permeability of planar membranes or films can be adjusted reversibly without permanently destroying the structures.

According to the invention it has been found that the forces applied by an external field can be tuned to achieve a desired permeability reversibly, while preserving the membrane's or film's integrity. Thus, it is possible to control membranes or films non-destructively. In particular, the permeability of a membrane or film can be increased by applying external forces and subsequently be returned to its initial state.

Membranes, in particular, are structures which, while in an open state, allow molecules to pass through. In particular, membranes separate two or more compartments. In films, in particular, molecules can go in and out on the same side. In particular, films are located on a substrate, preferably on a non-transparent substrate.

Nanoparticles which constitute absorption centers embedded into membranes or films such as, for example, into shells of microcapsules or into films can be used to control membrane or film permeability. The control can be performed by external fields, preferably by electromagnetic, optical or magnetic fields. The nanoparticles then serve as active centers which absorb the external energy. Nanoparticles being embedded into membranes or films such as into a shell or film are heated by the uptake of the external energy. The heated nanoparticles then, in turn, heat the nearby structures of the shell or the film. Thereby the network of the structure is transiently loosened and the permeability is reversibly changed.

By tuning the external energy, in particular, the time and/or the degree of permeability change of a membrane or film can be adjusted. According to the invention, the change of permeability of a membrane or film is transiently effected, in particular, a membrane or film is transiently opened for molecules having a predetermined size and thereafter again closed. When an external field is applied, the nanoparticles absorb energy and convert it into heat which dissipates the membrane or film network around the nanoparticles. When heated, the membrane or film network, in particular, a crystalline membrane or film network, becomes fluid-like. Thereby, the permeability of the membrane or film is increased. If application of external forces is arrested, the nanoparticles stop producing heat and the membrane or film structure around the nanoparticles rapidly cools, returning to its original less permeable and, in particular, crystalline state. To implement reversibility, preferably, the temperature on the membrane or film is raised locally by external force-nanoparticle interaction just above the glass transition temperature (T₉) of the membrane or film. In particular, the temperature is locally raised at least 1 , more preferably at least 2, in particular, at least 3 and, more preferably, at least 5 ⁰C above the glass transition temperature, but not higher than 20, preferably less than 10 and more preferably, less than 8 ⁰C above T₉.

Thus, according to the invention, membrane or film permeability can be reversibly changed. In particular, permeability is increased by applying an external field to the membrane or film. The membrane's or film's permeability then decreases again after the external field is removed or turned off and can again be increased subsequently when the external field is turned on again. Heat produced by absorbing nanoparticles transiently melts the membrane or film network around the nanoparticles, locally increasing membrane or film permeability. Switching off the external field brings the temperature of the molten membrane or film again below T₉, forcing the membrane or film to seal itself. - A -

The membranes or films can be spherical and be part of capsules such as micro- or nanocapsules and, in particular, form the shell of nano- or microcapsules, or can be planar membranes or films. Preferably, the membrane or film is a layer-by-layer membrane or film, a polyelectrolyte membrane or film, a polymer membrane or film, a lipid membrane or film or a cell membrane or film. Preferred are membranes or films comprised of polymeric, polyelectrolyte multilayer, lipidic and/or biodegradable materials and/or membranes or films of living cells. In a particularly preferred embodiment, the membrane or film comprises polyelectrolytes as building blocks. A polyelectrolyte/nanoparticle composite membrane or film is particularly preferred.

In another preferred embodiment, the membrane or film is a polymeric membrane or film, in particular, comprising biocompatible and/or biodegradable polymers.

In a preferred embodiment, a change of permeability can additionally be effected by the incorporation of biocompatible and/or biodegradable materials, in particular, biodegradable polymers, in the membrane or film.

The invention basically can be carried out with any vehicles having membranes or films, e.g. with drug delivery vesicles such as liposomes, red blood cells, micelles, microgels or other capsule structures. A preferred embodiment of the shell or film is a polymeric microcapsule or a polymeric planar membrane or film and, in particular, a polyelectrolyte multilayer.

The membranes or films can have a thickness ranging from the nanometer to micrometer to submillimeter range. The membranes or films, in particular, have a thickness of at least 1 nm, preferably at least 100 nm, more preferably at least 1 μm and more preferred at least 100 μm, and up to 1 mm, preferably up to 10 μm, preferred up to 1 μm. The membranes or films can encompass other molecules, in particular, active agents, medicaments, pharmaceutical agents and/or drugs.

According to the invention the term ,,nanoparticles" e.g. comprises nanoparticles and nanorods. The term ,,nanoparticles" also comprises active centers, in particular, absorbing centers, which can be used according to the invention as well in order to modify the permeability of membranes or films.

Such active centers e.g. are absorbing polymers or absorbing dyes. In a preferred embodiment, the nanoparticles are in the form of nanoparticle aggregates. Further colloidal particles covered or functionalized with nanoparticles can be employed.

The nanoparticles themselves are particles having a size of from 1 to 800 nm, preferably from 5 to 500 nm, more preferably from 10 to 100 nm and most preferably from 15 to 50 nm.

The nanoparticles can be metallic or non-metallic nanoparticles. Examples of suitable materials are organic materials and, in particular, organic dye materials. Absorbing polymer nanoparticles can also be used. Preferably, the nanoparticles are metallic nanoparticles and, in particular, noble metal nanoparticles. Most preferred are nanoparticles comprised of gold, silver and/or copper. It is also possible to use magnetic nanoparticles. Nanoparticle aggregates containing from 4 to 1000 nanoparticles are preferably comprised in the membranes or films. In a preferred embodiment, the nanoparticles are employed in relatively low concentration such as a surface coverage < 40%, in particular, < 20%, more preferably < 10%.

On the nanoparticles embedded in the membrane or film, an external force is exerted, in particular, an optical, an electromagnetic, an electrical, an ultrasound and/or a magnetic field. The irradiation of light is particularly preferred. However, also other external forces such as ultrasound can be applied. Preferably, remote control of permeability is effected. As external field, preferrably light is used having e.g. wavelengths in the IR or near-IR part of the spectrum, in particular, having wavelengths from > 600 nm, more particularly > 700 nm, more preferably > 800 nm and up to 1500 nm, preferably up to 1000 nm and more preferably up to 900 nm. IR light is particularly important in biotechnology due to low absorption of biological materials in said spectral window. However, it is also possible to use light of other wavelengths such as from 200 nm to 600 nm.

Currently, there is a wide use of electroporation for controlling permeability of living cells and other membranes or films. In a preferred embodiment, the invention relates to an optical and/or magnetic analog of electroporation. The advantage of the present invention is an unprecedented precision.

Irradiation of light is especially preferably effected by means of a laser so that light of specific wavelengths can be irradiated. One can use pulsed lasers or continuous wave lasers.

Nanoparticles absorbing in the near-IR part are most preferred according to the invention. Such an absorption can be obtained by using a suitable shape of the nanoparticles, e.g. nanorods, or by their aggregation state. According to the invention it is especially preferred to employ nanoparticles in the form of nanoparticle aggregates. Such aggregates, in particular, contain at least 4, in particular, at least 5, preferably at least 10, more preferably at least 20 and up to 1000, in particular, up to 200, more preferably up to 100 nanoparticles. Such structures preferably have an absorption peak between 700 nm and 900 nm. Gold nanoparticle aggregates are particularly preferred.

As indicated, a change in absorption properties can be achieved by forming nanoparticle aggregates. Nanoparticle aggregates can be formed e.g. by adding a salt, in particular, a salt comprising Na⁺ or K⁺ ions such as NaCI or KCI to nanoparticles. By adding salt, it is also possible to provide a non- uniform distribution of nanoparticles in a membrane or film.

The time of effecting a change in permeability properties of the membrane or film can be adjusted according to the invention by the time of exerting an external field. Preferably, the external field is applied for at least 1 second, preferably, for at least 2 seconds and, more preferably, for at least 5 seconds. While the external force can be applied as long as desired, it is applied preferably up to 5 minutes, more preferably, up to 1 minute and, most preferably, up to 20 seconds. The degree of permeability change depends on the energy applied with the external field. Preferably, an amount of energy is provided so that the membrane or film is transiently opened for the passage of molecules of a predetermined size, and after a predetermined period of time the membrane or film is again closed for these molecules having a predetermined size. For example, energy can be adjusted in such a way that molecules having a size up to 10 kDa, more preferably, up to 5 kDa, can pass through the membrane or film while larger larger molecules such as molecules having a size of e.g. 70 kDa are retained.

According to the invention, in particular, the permeability of the membranes or films is reversibly controlled. In particular, the application of external forces has a transient effect on the membranes or films. Controlled permeability changes of membranes or films, in particular, of planar membranes or films can be used e.g. for the separation of molecules. According to the invention it is thereby possible to adjust the separation size by tuning the external field applied. For example, the separation size can be adjusted to 5 kDa, 10 kDa or any other value. Separation of molecules is a large application area. Additional functionality can be gained by adding an electric field for facilitating the movement of molecules through the membrane or film, while gels and/or beads could be used for preliminary separation of molecules. Also a thermal gradient can serve as an additional mechanism for molecule separation. The control of membrane or film permeability and, in particular, remote control of membrane or film permeability can also be used for drug delivery, in particular, for drug delivery vehicles such as liposomes, red blood cells, gels, microgels, microcapsules, micelles, etc. By incorporating nanoparticles in the walls of the delivery vehicles controlled release of the incorporated drug can be achieved.

The inventive method allows numerous applications and possibilities. For example, it is possible due to the inventive method to allow size-dependent transient release of molecules from the interior of capsules or transient filling of the interior of capsules with molecules of different size. The size of the transiently opened pores thereby is controlled by the strength of the external field. Using a microfluidic-based setup allows for incorporation of microcapsules into bioreactors or living cells.

The method according to the invention also can be used for fusing vehicles. Thereby, an external field is applied and, by increasing the permeability of the membranes or films of two or more vehicles, these vehicles are fused together. By turning off the external field, the fusing becomes permanent.

In particular, capsules such as micro- or nanocapsules, vesicles or other drug delivery vehicles and bioreactors can be fused. The principles mentioned herein can also be applied to intracellular operations.

The inventive method can be used, in particular, for passing molecules through the membrane or film. Thereby, the external field is adjusted such that molecules having a predetermined size can pass through the membrane or film.

It is also possible to use the inventive method to release molecules which are embedded in the membrane or film. In particular, molecules or materials embedded in the membranes or films of micro- or nanocapsules or in planar membranes or films or biocompatible films can be released. The inventive concept of transient size-dependent pore opening can also be applied to planar membranes or films. Also in this case, the size of the transiently opened pores is controlled by the strength of the external field.

In a particularly interesting application, materials or molecules such as DNAs, proteins etc. located on the surface of films or membranes or incorporated inside films or membranes can be released. This application can be used, in particular, for transfecting cells with DNA.

According to the invention it has been found that by doping membranes or films with nanoparticles and, in particular, with nanoparticle aggregates a minimum power of an external field such as minimum laser power is required to reversibly open the membranes or films (i.e. control their permeability). A focused laser beam of incident power as low as 10 mW up to 100 mW, preferably 20 mW up to 80 mW, is sufficient to open the membranes or films, e.g. of capsules without causing deformation, destruction or explosion of the membranes or films or microcontainers. A cw-laser light is preferably applied to the material for 1 s to 20 s, in particular, from 2s to 10 s. Also, a pulsed laser source can be employed such as a pulsed femtosecond, picosecond or nanosecond laser in a range from 1 μJoule to 10 Joule.

Upon illumination, the temperature of the nanoparticles rises because light energy or another external energy exerted is converted into heat. The heat produced by the nanoparticles upon light absorption induces the temperature of the membrane or film material surrounding the nanoparticles which act as absorption centers to rise. The temperature rise results in melting of regions of the membrane or film so that a change in wall permeability occurs. In case of a polyelectrolyte multilayer membrane or film a temperature increase around a nanoparticle sufficient to exceed the glass transition (T₉) of the surrounding polyelectrolytes is sufficient to produce molten regions which are more permeable. Thus, encapsulated material can be released or empty capsules or cells can be loaded or molecules can be separated through planar membranes or films due to their size. In a preferred embodiment of the invention, the membrane or film is a polymeric membrane or film, in particular, a multilayer polyelectrolyte membrane or film. In a further preferred embodiment, nanoparticle aggregates, in particular, Au nanoparticle aggregates are used. In particular, release of encapsulated materials from capsules containing aggregates of nanoparticles such as gold particles can be performed at low power of near IR laser, while under similar conditions no release takes place for capsules containing non-aggregated gold particles.

In a further preferred embodiment, membranes or films are used which have a low Tg such as a T₉ between 35 ⁰C and 45 ⁰C, in particular, between 40 ⁰C and 45 °C. Those systems can be used for in vivo studies as typical cell experiments are conducted at 37 ⁰C. Therefore, in the presence of living cells, one needs a rise in temperature of only 3 ⁰C to effect a change in permeability of such a polymeric shell. Nanoparticle aggregates, in particular, gold nanoparticle aggregates heated with an infrared laser beam can easily fulfill this requirement. Most preferably, the polyelectrolyte multilayer capsule comprises layers of PDADMAC and of PSS.

In a further preferred embodiment of the invention, vehicles comprising a membrane or film are delivered into cells prior to reversibly controlling the permeability of the vehicle membrane or film. Delivery is preferably performed with a microfluidic device.

The invention further relates to a microfluidic based intracellular delivery and release system. External control of permeability of membranes or films and, in particular, of microcapsules as well as nanoparticle-based sensors as described below can be used in microfluidic based intracellular drug delivery systems. Such a system uses microfluidics channels for delivery of vehicles functionalized with absorbing nanoparticles or absorbing dye molecules, for example, photodynamic therapy agents, absorbing dyes, etc. or magnetic particles. The microcapsules can then be electroporated or injected into cells, while the release takes place remotely by an external field such as a laser, a magnetic field or MRI. As delivery vehicles, e.g. microcapsules, in particular, polymeric microcapsules, liposomes, microgels, red blood cells, micelles or other drug delivery vehicles can be used. The described system can be employed for intracellular release of encapsulated materials applicable for testing drugs on a cellular level. Thereby, capsules having absorbing centers such as nanoparticles in their shell are remotely activated.

Thus, the method according to the invention can be employed for controlled delivery, electro-optical incorporation and remote release of delivery vehicles into cells, in particular, into living cells. For performing this embodiment, preferably a setup equipped with microfluidic channels, an electroporation device and a laser for intracellular delivery and release is provided.

Preferably, capsules with a low concentration of absorbing centers in the shell are used to allow for controllable release, which leads to a non- disruptive intracellular release of drugs from the microcapsules which can be used for studying the effects of a drug, etc. on living cells.

However, it is also possible to use capsules having a high density of absorbing centers. The high temperatures resulting from conversion of external energy into heat by the absorbing centers, which high temperatures exceed the viability threshold, can be used for thermo-ablation.

The intracellular delivery and release system described above can also be implemented in a high-troughput device, e.g. for studying intracellular processes. For high-throughput operation, for example, a spatial light modulator (SLM) is inserted in the optical path of the laser beam. A SLM allows to direct the optical beam onto designated places on a high- throughput chip. Therefore, multiple sites on a high-throughput chip can remotely activated and read out. The laser light can be brought onto a cellular chip in free space, through a microscope objective or through an optical filter. Continuous wave or pulsed wave lasers can be used. Further, super-resolution methods can be applied to trace the effects of released substances from delivery vehicles inside living cells. Also, conformation and/ or configuration of molecules such as polymers, proteins, etc. can be studied using said system. Also, knocking down specific genes by siRNA can be performed remotely on a chip in a single cell or using a high-throughput system. It is also possible to connect a spectrometer in a filter-optical configuration.

For high-throughput operation it is also possible to employ a setup, in which cells are flowing and are activated when they pass an externel energy source such as a laser.

However, it is also possible for high-throughput systems to use another external energy source such as a magnetic field or ultrasound. In this embodiment, drug delivery vehicles are functionalized with corresponding absorbing centers.

In a further embodiment of the invention, drug delivery vehicles are functionalized with remote release capabilities for in vivo applications. The delivery of the external energy can then be performed by optical fibers. Enhancement by PDT (photodynamic therapy) is also possible. In case of magnetic remote release, MRI (magnetic resonance technics) can be used.

The invention further concerns a method for locally affecting a substrate material by interaction of particles with external forces. In this application, the sizes of particles can be within a wide range from nanometer to microns and higher. Preferably, however, also in this application, nanoparticles are employed. By exerting external forces to the particles, they heat and sink into the molten substrate material. Using this method, Janus particles and, in particular, Janus nanoparticles can be produced. Preferably, nanoparticles are deposited on a surface of a material, which nanoparticles have functional groups. Exposing the particles to an external field to yield direct or indirect heat results in sinking of the nanoparticles into the material, while the depth of sinking or embedding can be controlled by the heat supplied and the thickness of the substrate material as well as the thermal properties of the substrate material. Indirect heat can be applied e.g. by applying external fields such as electromagnetic fields, for example, optical, electrical or magnetic fields. By energy uptake the particles are heated, which is termed indirect heating herein.

The non-embedded functional groups are then exposed and can be exchanged for other functional groups. Removing the sacrificial template material results in Janus nanoparticles.

The invention, therefore, can be used for producing Janus particles, comprising the steps of: depositing particles on a surface of a material, exposing the particles to an external field such as direct or indirect heat such that the particles at least partly sink into the material, modifying the surface of the particles which is not embedded in the material, and removing the material.

Further, it is possible according to the invention to probe thermal properties of materials. Properties, in particular, thermal properties of materials can be tested by depositing nanoparticles on the surface to be investigated. Then, the properties of the material, in particular, thermal properties, can be probed by exerting external energy on the nanoparticles. Material thermal properties can be probed e.g. by AFM measurements after depositing nanoparticles on the surface or after embedding nanoparticles inside the material to be investigated. The AFM measurements provide information about the material influenced by thermal or local thermal effects.

The invention, therefore, can be used for probing properties of materials, in particular, for probing surface and/or interface properties of materials by attaching particles, in particular, nanoparticles to the material. Such a method preferably comprises the steps of depositing particles, in particular, nanoparticles on the material to be investigated, and probing properties of the material, in particular, thermal properties.

The technique described above can also be used for nano-imprinting. Preferably, in a first step, nanoparticles are deposited or self-assembled onto a surface of a material. The materia! can be hydrophobic or hydrophilic. Upon exposure of this structure to external energy by an external field such as laser light, the nanoparticles are immersed into the substrate. The contact angle of the hydrophobic or hydrophilic surface determines how the nanoparticles are immersed. The protrusions created by nanoparticles can be utilized as nanopatterned template. Further, it is possible to dissolve or remove the supporting template and to extract a nanopattem structure. In all applications described it is important to control the distribution of absorbing centers, in particular, nanoparticles on the surface. Such distribution control is important to ensure confinement of absorbed energy.

The invention further provides surfaces with controllable elasticity. To obtain such surfaces, nanoparticles are embedded into a material such as a film, in particular, polymeric film, or a bioactive surface. After applying an external field such as laser illumination the elasticity or softness of the surface changes. This method can be applied to directed living cell growth on a predefined pattern. Patterning on a large scale can be controlled, for example, by SLM. Temperature distributions can be controlled e.g. via intensity or via distribution of nanoparticles. The properties of the surface can be controlled transiently by applying an external field.

The invention also relates to a surface with controllable elasticity and relase capabilities. According to the invention the release of a drug or active substances can be performed remotely within a film. To this end, microcapsules, liposomes, red blood cells, etc. comprising nanoparticles can be embedded in the film. After applying an external field such as laser illumination the delivery vehicles exposed to the field such as laser light release their content. This can be used e.g. in biological applications such as releasing drugs, medicine, etc. upon application of an external field. It can also be used, however, to affect or protect the surfaces.

This remote release can be combined with elasticity control. Further, nanoparticles directly embedded into the film can be used for releasing contents of microcontainers such as liposomes, microcapsules, etc. which are not functionalized with absorbing nanoparticles. Further, it is possible to release drugs from liposomes by adding metallic absorbing nanoparticles and exposing the liposomes comprising nanoparticles to laser light.

Both for elasticity and elasticity with release capability it is important to control the distribution of nanoparticles. A uniform distribution at low concentration such as low surface filling factor < 0.05 is obtained by just adding nanoparticles. Non-uniform distribution of nanoparticles is obtained by adding a salt, for example, NaCI or KCI. In case of high concentration of nanoparticles, e.g. a surface filling factor > 0.20, direct absorption leads to non-uniform distribution of nanoparticles, while addition of polymers leads to uniform distribution of nanoparticles.

Controlled distribution can also be applied to control the interface of electron transfer complexes in solar and fuel cells. In these applications, the interface between the electron donors and electron acceptors can be controlled, thus increasing efficiency of operation.

The invention further relates to fusion of microcontainers such as cells, liposomes or microcapsules. Fusion of cells and delivery vehicles is an important process. For fusing, the microcontainers, e.g. cells, are brought into contact. Preferably, the cells are manipulated e.g. through optical trapping by a laser beam to bring them into contact. In a first embodiment, it is possible to fuse microcapsules just by bringing them into contact. However, using microcontainers or microcapsules functionalized with absorbing nanoparticles facilitates the fusion process upon subjecting to an external field. During fusion, microcapsules interact and form a new entity sharing the same membrane or film. Contents of microcapsules can also be released controllably. An interesting application of this technique is the fusion of cells.

Further, it was found that single nanoparticles and nanoparticle aggregates absorb visible and near-IR light very differently. Therefore, application of an external field, for example, an electromagnetic, magnetic or optical field, on single nanoparticles and on nanoparticle aggregates in mass spectrometry will lead to differentiation of signals of molecules such as proteins or peptides. This results in a method for detection of molecules, in particular, biomolecuies, using mass spectroscopy, comprising attaching nanoparticles and/or nanoparticle aggregates to the molecules and exerting an external field on the molecules. Further, single nanoparticle and nanoparticle aggregates amplify Raman signal in a different way. Therefore, single nanoparticles and nanoparticle aggregates can be used for Raman signal amplification and also to pump-probe Raman signals. This results in a method for detection of molecules, using Raman spectroscopy, comprising attaching nanoparticles and/or nanoparticle aggregates to the molecules and exerting an external field, and, in particular, in a method for pump-probe enhancement of Raman signals, comprising attaching nanoparticles and/or nanoparticle aggregates to the molecules and applying an external field.

The principle of the invention also can be employed for structure determinations. The invention, therefore, also relates to a nanoparticle- based sensor and, in particular, to a method of structure determination of molecules, in particular, of biomolecuies, wherein the molecules are functionalized with at least one nanoparticle.

Nanoparticles, in particular, absorbing nanoparticles, can also be used for sensor applications for the detection of molecular configuration. In this application, the structure, configuration and/or aggregation state of molecules, in particular, biomolecules such as proteins can be determined using absorbance characteristics of nanoparticles by functionalizing the molecules with at least one nanoparticle. In typical conventional experiments for protein structure determination FRET is used to detect the configuration of proteins. Proteins labeled with multiple fluorescent FRET particles, for example, quantum dot particles, allow for precise structure determination. It has now been found that multiple absorbing nanoparticles allow for structure determination due to the absorption of closely located nanoparticles which form aggregates due to their vicinity. The configuration of molecules can be determined, since non-aggregated single nanoparticles show different absorption spectra than aggregated nanoparticles. For example, single Au nanoparticles show a single absorption peak at about 520 nm, while absorption is shifted in Au nanoparticle aggregates to near-IR (700 nm to 900 nm). Distinguishing nanoparticles and/or nanoparticle aggregates optically allows to detect nanoparticles and structures with nanoparticles spectroscopically. Thus, the configuration of molecules can be measured by attaching nanoparticles to the molecules and determining absorption.

Further, according to the invention, the conformation and/or configuration of molecules, in particular, of biomolecules such as proteins and peptides functionalized with nanoparticles can be studied, affected and/or changed by exerting external forces which result in heating produced by the nanoparticles.

The invention is further illustrated by the Figures and Examples as given below.

Fig. 1 shows a controllable permeability change of polymeric microcapsules and layers. Microcapsules having a shell composed of polyelectrolyte multilayers functionalized with Au nanoparticles were exposed to laser light. Encapsulated material being dextran polymer having a molecular weight of about 10 kDa could be released in a controllable way. Fig. 1A shows that high concentration of nanoparticles on microcapsules with a surface cover > 50% and an Au nanoparticle size > 40 nm results in destruction of the shell.

Fig. 1 B shows that by lowering the concentration to a surface coverage < 20% using Au nanoparticles having a size of 20 nm results in release of encapsulated materials without destruction of the microcapsule. Exposing a similar capsule to lower irradiation intensity by bringing the shell of the microcapsule into the center of the laser beam leads to a controllable partial release as shown in Figs. 1C-E. Control of time of the release is shown in Fig. 1 F. Molecular weight differentiation can also be implemented as shown in Fig. 1 G.

Fig. 2 shows a comparison between single gold nanoparticles and gold nanoparticle aggregates.

As shown in Fig. 2A, a solution of citrate-stabilized gold nanoparticles has a red color as seen through a plastic cuvette. Its corresponding UV/visible absorbance spectrum shows a strong absorbance peak at 520 nm (Fig. 2B). Fig. 2C shows a TEM image of dry (PDADMC/Au/PSS)₄ shells with non- aggregated gold particles.

The color of a gold nanoparticle aggregate solution formed by adding salt is blue/grey as shown in Fig. 2D. The corresponding absorption spectrum is shown in Fig. 2E. Fig. 2F shows the general appearance of these aggregates as seen when inserted in the wall of a (PDADMAC/Au/PSS)4 microcapsule in the TEM image. Scale bars in the TEM images correspond to 500 nm.

Fig. 3 shows release properties of capsules containing non-aggregated gold particles or aggregated gold particles, respectively.

Fig. 3, top, shows LSCM images of microcapsules with encapsulated Alexa Fluor 555 dextran before (Fig. 3A) and after (Fig. 3B) exposure to an infrared iaser (830 nm). No capsules were opened at any power intensity used as shown in Fig. 3C.

Fig. 3, bottom, shows LSCM images of capsules comprising aggregated gold particles with encapsulated labeled dextran before (Fig. 3D) and after (Fig. 3E) exposure to an infrared laser (830 nm) using an incident intensity of 65 mW. Fig. 3F shows a plot correlating the percent of capsules opened (30 capsules per measurement) as a function of power (solid line) and its derivative (dashed line).

Fig. 4 shows detection of molecular configuration of proteins.

Fig. 4A shows conventional FRET-based detection. Fig. 4B shows multiparticle absorption-based detection.

Fig. 5 shows absorption spectra of non-aggregated nanoparticles (red) and controllably aggregated nanoparticles (blue) taken after 3 min of adding NaCI to Au nanoparticles. While non-aggregated Au nanoparticles exhibit a single peak at 520 nm, aggregated Au nanoparticles show a near-IR absorption peak.

Fig. 6 shows a microfluidic-based intracellular delivery system which can be used for studying intracellular processes.

Fig. 7 shows the principle of intracellular release. Fig. 7(A) shows remote activation of capsules with high density of absorbing centers resulting in high temperatures. Fig. 7(B) shows controllable release using microcapsules having shells comprising a low concentration of absorbing centers resulting in non-disruptive intracellular release.

Fig. 8 shows a high-throughput system including a spatial light modulator inserted into the optical path of the setup outlined in Fig. 6. Fig. 9 shows the principles of embedding nanoparticles into polymers (Fig. 9A-D). An AFM image and profile of a nanoparticle before (Fig. 9A1 ) and after (Fig. 9D1 ) illuminating the nanoparticles located on a polyelectrolyte multilayer film is also shown. Figs. 9A2 and 9D2 show profiles of the same nanoparticle before and after illumination with 150 mW.

Fig. 10 shows Janus nanoparticles having different functionalities on their surfaces.

Fig. 11 shows probing of thermal properties of materials by AFM. An AFM can be performed after depositing nanoparticles on the surface (Fig. 11 A) or after embedding nanoparticles inside the material (Fig. 11 B).

Fig. 12 shows a theoretical simulation of a nanopattemed surface. First nanoparticles are deposited or self-assembled on a surface. Upon exposure of this structure to laser light, the nanoparticles are immersed into the substrate.

Fig. 13 shows remote control of the softness or elasticity of a surface upon laser illumination. Fig. 13A shows a material having embedded nanoparticles. Fig. 13B shows the change in relative softness of this material due to laser illumination.

Fig. 14 shows the temperature distribution around nanoparticles after laser illumination. The temperature distribution can be controlled via intensity (Fig. 14A) or via distribution of nanoparticles (Fig. 14B).

Fig. 15 shows modeling of temperature distributions for non-aggregated (top) and aggregated nanoparticles (bottom) illuminated with a near-IR laser (830 nm). Non-aggregated Au nanoparticles do not possess an absorption in the near-IR part of the spectrum. For 20 nm Au nanoparticles, the absorption coefficient is about 0.02, so the temperature rise at 50 mW of incident power is less than 1 K (Fig. 15A). For a single line of 4 aggregated nanoparticles, a temperature rise of 7 K can be produced (Fig. 15B). TEM images of uniform distribution and aggregates of nanoparticles are also shown. The scale bars for the TEM images are 100 nm.

Fig. 16 shows the release of encapsulated materials in a film.

Fig. 16A shows microcontainers such as microcapsules, liposomes or red blood cells comprising nanoparticles being embedded in a film. Fig. 16B shows that upon laser illumination the delivery vehicles release their content. Figs. 16C and D show that remote release can be combined together with elasticity control and that nanoparticles directly embedded into a film can be used for releasing the contents of microcontainers which are not functionalized with absorbing nanoparticles.

Fig. 17 shows remote release of encapsulated materials from liposomes. Metallic absorbing nanoparticles are added to liposomes and these liposomes were then exposed to laser light. Fig. 17, left, shows the fluorescent image before release, Fig. 17, middle, a transmission microscope image and Fig. 17, right, the fluorescent image after release.

Fig. 18 shows TEM images of uniform (A) and non-uniform (B) distributions of nanoparticles at low concentrations. Figs. 18 (C) and 18 (D) show TEM images of non-uniform and uniform distributions at high concentration of nanoparticles.

Fig. 19 shows fluorescence microscopy images of capsules before (Fig. 19A), during (Fig. 19B) and after (Fig. 19C) fusion of microcapsules. The scale bar corresponds to 3 μm. The insert shows microcapsules separated before fusion.

Fig. 20 shows remote release from microcapsules: (A) schematics of nanoparticle functionalized polymeric nanomembranes or nanofilms opening channels upon laser illumination; (B) a polymeric microcapsule shell acts as a reversible nanomembrane or nanofilm. Upon laser light illumination the microcapsule (left image)partially releases encapsulated polymers and reseals (middle). After the second illumination the microcapsule completely releases its content (right). Profiles in the left upper corner are drawn along the green line. Scale bars correspond to 5 μm.

Examples

Example 1 : Preparation of aggregates of Au nanoparticles

A solution of citrate-stabilized AuNPs having an average diameter of 20 nm was provided. Citrate molecules added as a coordinating agent during the preparation of AuNPs prevent aggregation of the nanoparticles with each other through electrostatic stabilization.

Then, this solution is mixed with an equal volume of a solution of NaCI (0.1 M). Thereby, aggregation proceeds and the color rapidly changes from ruby/ red to blue/grey (cf. Fig. 2). Aggregation occurs, since the stabilizing charges of the citrate molecules covering the gold nanoparticles are compensated by a sufficient number of oppositely charged ions that lower the long-range electrostatic repulsion and allows short-range attractive forces such as van der Waals forces to dominate.

The absorbance spectrum is shifted from λ_max = 520 nm to a novel broad absorption region between 700 and 900 nm with a maximum centered at about 740 nm.

A similar absorption peak, red-shifted, appears upon mixing a volume citrate- stabilized AuNPs with an equal volume of a potassium chloride solution (0.1 M).

Example 2: Formation of microcapsules comprising nanoparticles in the shell Polymeric microcapsules were fabricated by alternatively coating SiO₂ templates having an average diameter of 4.78 μm with poly(diallyldimethylammonium chloride) (PDADMAC) (2 mg/ml, 0.5 M NaCI), 20 nm colloidal gold and poly(styrene sulfonate) (PSS) (2 mg/ml, 0.5 M NaCI). At the end of the shell assembly, the coated silica particles were treated with hydrofluoric acid (0.3 M) and thereafter thoroughly rinsed with water to remove the acid. Hollow capsules having an average diameter of 4.8 μm and possessing the shell structure (PDADMAC/Au/PSS)₄ were obtained. To produce shells having single nanoparticles incorporated therein, citrate-stabilized AuNPs were employed as colloidal gold.

To build up shells containing aggregated gold NPs equal volumes of colloidal gold solution (citrate-stabilized) and 0.1 M NaCI were gently mixed for 60 sec, forming gold nanoparticle aggregates. This solution of gold nanoparticle aggregates was added to a solution containing silica particles (average diameter 4.78 μm) coated with PDADMAC as outermost layer. Thereafter, a solution of PSS was applied. After treating the particles four times with PDADMAC, aggregated gold nanoparticles and PSS the coated silica particles were treated with hydrofluoric acid (0.3 M) and thoroughly rinsed with water to remove the acid. Hollow capsules having an average diameter of 4.8 μm and possessing the shell structure (PDADMAC/Au_aggregates/PSS)₄ were obtained.

Electron microscopy revealed the presence of mesh-like aggregates containing between 20 and 100 gold NPs within the capsule wall.

Encapsulation of Alexa Fluor 555 dextran (10 kDa) was achieved by heating a mixture of capsules and dextran (0.1 mg/ml) at 54 ⁰C for 20 min.

Example 3: Remote release

Microcapsules having the shell structure (PDADMAC/Au/PSS)₄ were used to compare the release efficiency of shells containing non-aggregated AuNPs or aggregated AuNPs. For laser-induced release experiments using an IR laser (830 nm), fluorescently labeled dextran (10 kDa) was encapsulated. Permeability studies showed that the samples retained the encapsulated material and no changes in fluorescence intensity was found after 5 months of storage (4 ⁰C, dark conditions). (PDADMAC/Au/PSS)₄ microshells with encapsulated dextran were then irradiated using a IR laser. 30 capsules were irradiated in each experiment. The release of encapsulated materials from the microcontainers containing non-aggregated and aggregated AuNPs was remarkably different. Capsules containing non-aggregated AuNPs exposed to an IR laser beam were not opened (cf. Fig. 3A, B) within the whole range of incident laser powers (1-90 mW), also indicating that no photobleaching takes place. The fluorescence of laser irradiated unopened microcapsules containing non-aggregated AuNPs did not change after 48 h. A graph plotted using the percentage of opened capsule as a function of laser intensity gives a flat line (Fig. 3C).

In sharp contrast, the dextran content encapsulated in microcapsules with aggregated nanoparticles was entirely released at about 70 mW. Each capsule was irradiated with the infrared laser for 5 sec at 65 mW and thereby opened, as shown in Figs. 3D and 3E. The capsule that remains fluorescent in Fig. 3E after irradiation was deliberately kept away from the laser to demonstrate that the fluorescence intensity of the capsules did not change after irradiation proximal capsules. The percentage of microcapsules optically opened in this manner decreased at lower intensities as illustrated in Fig. 7F (solid line). The minimum laser power required to remotely open capsules containing aggregates AuNPs was found to be about 10 mW. At intensities below 60 mW a second laser exposure of 10 sec was performed on microshells that were not opened in the first experiment. Thereby, up to 12% more capsules could be opened after the second irradiation. A derivative plot of the data points found in Fig. 7F (solid line) is also provided (Fig. 7F, dashed line). The peak of this curve centered at 36 mW is referred to as the threshold intensity and is defined as the intensity necessary to open more than 50% of capsules.

The LSCM images in Fig. 3 demonstrate that upon irradiation the capsules released their fluorescence content entirely. In addition, the laser-induced release of material from microcapsules was never accompanied by their deformation or ..explosion". Thus, a non-destructive release is given.

Example 4: Heat distribution around nanoparticle assemblies

As shown, release of encapsulated materials from capsules containing aggregates of gold particles can be performed at low power of near-IR laser, while no release takes place for capsules containing non-aggregated gold particles. This difference is due to a more efficient, localized temperature rise around aggregates of gold nanoparticles due to a higher absorption coefficient at the irradiated wavelengths and a higher surface to volume ratio. The high release efficiency of capsules containing aggregates of AuNP is at least partly due to the localization of the particles over a small area of the microcapsule wall. Concentration of the particles to a small volume within the capsule shell leads to localization of the heat produced by the nanoparticles upon light absorption. Therefore, aggregating AuNPs increases the surface to volume ratio of the IR-absorbing nanoparticles, inducing the temperature of the shell material surrounding the absorption centers to rise. Thus, non-aggregated particles have the double disadvantage of not absorbing much in the IR range and having to absorb more light to produce enough heat to melt sufficiently thick regions of the polyelectrolyte shell complex so that a change in wall permeability could occur. Therefore, microcapsules containing non-aggregated AuNPs require relatively insense laser irradiation at a suitable wavelength, in particular, between 200 and 600 nm, which, in turn, is more likely to induce irreversible damages to the shell, its content and host material such as living tissues or cells.

In contrast thereto, a milder temperature increase around an aggregate of AuNPs is observed sufficient to exceed the glass transition (T₉) of the surrounding polyelectrolytes within the capsule shells. Being less dense above T₉, molten regions of the capsule shell become more permeable, allowing encapsulated material to leak out. Using differential scanning calorimetry, it was determined that (PDADMAC/PSS)₄ shells in water posses an endothermic peak centered at 40 °C, with an onset located at 35 ⁰C. Thus, for experiments done at ambient temperature of 25 ⁰C, only an increase of about 10 ⁰C is necessary to exceed T₉. For in vivo studies conducted at 37 ⁰C only a temperature rise of 3 ⁰C is needed to affect the permeability of the polymeric shell.

A simulation of temperature rise of non-aggregated and aggregated AuNPs is shown in Fig. 15. The size of nanoparticles taken was 20 nm, the absorption coefficient of nanoparticles, Q, is 1.6 and the surface plasmon resonance (520 nm) and Q is 0.02 at 830 nm. Using these parameters, the temperature rise on a single AuNP was calculated to be about 0.5 K when illuminated at 830 nm with a power of 50 mW. For a linear change of 4 AuNPs forming an aggregate the absorption coefficient at 830 nm is estimated to increase fivefold (Q = 0.1 ). Thereby, it was calculated that a single nanoparticle increases its immediate environment by less than 1 K, while a linear assembly of 4 nanoparticles can produce up to 7 K. Larger aggregates will produce even higher temperature rises.

Example 5: Reversibly permeable membranes or films of polymeric microcapsules

Polymeric microcapsules used as a model system with gold NPs (nanoparticles) were fabricated. 4.78 μm SiO2 templates were alternatively coated by poly(diallyldimethyl- ammonium chloride) (PDADMAC) (2 mg/mL, 0.5 M NaCI), 20 nm colloidal gold and poly(styrenesulfonate) (PSS, 2 mg/mL, 0.5 M NaCI). Gold Nps do not possess near-IR absorption, but a near-IR absorption peak can be formed by aggregating them by, for example, adding salt ions. The color of the gold solution changes from red to bluegray within seconds after adding salt resulting in the apparition of a near-IR absorption peak between 700 and 900 nm. To build shells containing aggregated gold NPs, equal volumes of colloidal gold solution and 0.1 M NaCI were gently mixed for 60 s and added to a solution containing the capsules templates. Then the cores (templates) were dissolved. The final capsules composition was (PDADMAC/Au/PSS)4. Electron microscopy revealed the presence of meshlike aggregates containing up to 100 gold NPs within the capsules wall.

The encapsulation of Alexa Fluor 555 dextran (10 kDa) was achieved by heating a mixture of capsules and dextran (0.1 mg/ml) at 54 ⁰C for 20 min. Remote release experiments were conducted using an IR laser.

Irradiating the capsules for 5 s or more was enough time to allow the entire capsule content to diffuse out. The minimum laser power required to remotely open capsules containing aggregated gold Nps was found to be 10 milliwatts. At 60 mW, 100% of the capsules had lost their load. In addition, the laser-induced release of material from the shell was never accompanied by deformation or "explosion" of the microcontainers. The light absorbed by metal NPs is converted into thermal energy. In this regard, the spectral features of aggregates of gold NPs are reminiscent of nanorod-like structures. Figure 5 presents absorption spectra of gold NPs (red curve) and aggregates of these Nps (blue curves). It can be seen from these data that NPs possess a single surface plasmon resonance peak situated around 520 nm and weak near-IR absorption. During salt induced NPs aggregation a second peak is formed in the IR region, and this second peak moves with time toward higher wavelengths. In this sense, the spectral features of aggregates of NPs are less versatile than those of nanorods but the preparation of aggregates is simpler and does not require specific surface- active compounds. Also, according to Figure 5, the position of the near-IR absorption peak can be controlled via the time elapsed from the initial aggregation till adsorption. The mechanism for controlling the permeability of nanomembranes or nanofilms can be understood by conducting an analysis of the interaction of laser light with NPs. Upon illumination, the temperature on the NPs rises because light energy is converted into heat. More efficient heat production can be obtained by controlling the distribution of NPs embedded within polymeric nanomembranes or nanofilms.

Furthermore, to implement reversibility one needs to locally raise temperature on the nanomembrane or nanofilm just above the glass transition temperature (Tg) of the polyelectrolyte complex. A mean Tg as low as 40 ⁰C was measured for this system with a high temperature phase distinguished by high elasticity and high permeability. The relatively low Tg of this polymeric system has major implications for in vivo studies as typical cell experiments are conducted at 37 ⁰C. Therefore, in the presence of living cells one needs a rise in temperature of only 3 ⁰C to affect the permeability of such a polymeric shell. Gold NPs heated with an infrared laser beam can easily fulfill this requirement.

Figure 2OA illustrates the schematics of a section of polyelectrolyte/NPs composite nanomembrane or nanofilm as would be found in a microcapsule with encapsulated material within its cavity. Upon IR laser illumination the NPs' aggregate generate heat and the encapsulated material (green) can diffuse through the membrane or film. By using aggregates of NPs the membrane or film becomes sensitive enough to IR irradiation, that it is possible not only to avoid destructive heat generation, but also to reversibly control membrane or film permeability. The magnified wall region on the right shows how this is possible. When the infrared laser is on, the NP absorbs light and converts it to heat, which dissipates to the polyelectrolyte network around it. When heated, the crystalline polymeric network becomes fluidlike allowing the release of encapsulated molecules through the membrane or film. If the irradiation is arrested, the NP stops producing heat and the polymeric complex around the NP rapidly cools, returning to its impermeable crystalline state.

Figure 2OB presents experimental evidence of reversibly permeable nanomembranes or nanofilms. Two capsules doped with aggregated gold NPs and filled with a fluorescent polymer can be seen. Fluorescence profiles are plotted from the green line for each image (inset). The upper microcapsule was exposed to the laser beam, whence its fluorescence decreased. Upon a second exposure, the fluorescence emitted by the capsule vanished, while the unexposed microcapsule below retained its original fluorescence intensity. Control experiments using microcapsules that lacked NPs showed no photobleaching. No change in fluorescence intensity occurred between the first and second laser exposure supporting that the membrane or film sealed itself after the laser was turned off and that the permeability changes observed after illumination are reversible.

Claims

1. A method for reversibly controlling permeability of a membrane or film comprising nanoparticles, wherein an external field is applied to the membrane or film.

2. The method according to claim 1 , wherein the nanoparticles are comprised in the membrane or film in the form of nanoparticle aggregates.

3. The method according to any of the preceding claims, wherein the external field is light, ultrasound and/or a magnetic field.

4. The method according to any of the preceding claims, wherein the external field is applied such that the membrane or film is not permanently destroyed.

5. The method according to any of the preceding claims, wherein the membrane or film is a layer-by-layer membrane or film, a polyelectrolyte membrane or film, a polymer membrane or film, a lipid membrane or film or a cell membrane or film.

6. The method according to any of the preceding claims, wherein the membrane or film is comprised of biocompatible and/or biodegradable polymers.

7. The method according to any of the preceding claims, wherein the membrane or film further comprises active agents, medicaments and/or drugs.

8. The method according to any of the preceding claims, being an optical and/or magnetic analog of electroporation.

9. The method according to any of the preceding claims, wherein the membrane or film is a planar membrane or film or a membrane or film of a microcapsule.

10. The method according to any of the preceding claims, wherein molecules are passed through the membrane or film.

11. The method according to any of the preceding claims, wherein molecules embedded in the membrane or film are released.

12. The method according to any of the preceding claims, wherein the permeability of the membrane or film is reversibly controlled such that the membrane or film is transiently opened for the passage of molecules of a predetermined size by applying an external field and, after a predetermined period of time, the membrane or film is again closed for these molecules having a predetermined size by turning off the external field.

13. The method according to any of the preceding claims, wherein the permeability of the membrane or film is remotely reversibly controlled.

14. The method according to any of the preceding claims, wherein molecules having different molecular weight are separated by reversibly opening a membrane or film.

15. The method according to any of the preceding claims, further comprising a step of delivering vehicles comprising a membrane or film into cells prior to reversibly controlling the permeability of the vehicle membrane or film.

16. The method according to claim 15, wherein the delivery is performed with a microfluidic device.