In an effort of developing clinical LANTCET (laser-activated nano-thermolysis as cell elimination technology) we
achieved selective destruction of individual tumor cells through laser generation of vapor microbubbles around clusters
of light absorbing gold nanorods (GNR) selectively formed in target tumor cells. Among all gold nanoparticles,
nanorods offer the highest optical absorption in the near-infrared. We applied covalent conjugates of gold nanorods with
targeting vectors such as monoclonal antibodies CD33 (specific for Acute Myeloid Leukemia), while GNR conjugates
with polyethylene-glycol (PEG) were used as nonspecific targeting control. GNR clusters were formed inside the tumor
cells at 37 °C due to endocytosis of large concentration of nanorods accumulated on the surface of tumor cells targeted at
4 °C. Formation of GNR clusters significantly reduces the threshold of tumor cell damage making LANTCET safe for
normal cells. Appearance of GNR clusters was verified directly with optical resonance scattering microscopy.
LANTCET was performed in vitro with living cells of (1) model myeloid K562 cells (CD33 positive), (2) primary
human bone marrow CD33-positive blast cells from patients diagnosed with acute myeloid leukemia. Laser-induced
microbubbles were generated and detected with a photothermal microscope equipped with a tunable Ti-Sa pulsed laser.
GNT cluster formation caused a 100-fold decrease in the threshold optical fluence for laser microbubble generation in
tumor cells compared with that in normal cells under the same targeting and irradiation conditions. Combining imaging
based on resonance optical scattering with photothermal imaging of microbubbles, we developed a method for detection,
image-guided treatment and monitoring of LANTCET. Pilot experiments were performed in flow mode bringing
LANTCET closer to reality of clinical procedure of purging tumor cells from bone marrow grafts.
LANTCET (laser-activated nano-thermolysis as cell elimination technology) was developed for selective detection and
destruction of individual tumor cells through generation of photothermal bubbles around clusters of light absorbing
gold nanoparticles (nanorods and nanoshells) that are selectively formed in target tumor cells. We have applied bare
nanoparticles and their conjugates with cell-specific vectors such as monoclonal antibodies CD33 (specific for Acute
Myeloid Leukemia) and C225 (specific for carcinoma cells that express epidermal growth factor -EGF). Clusters were
formed by using vector-receptor interactions with further clusterization of nanoparticles due to endocytosis. Formation
of clusters was verified directly with optical resonance scattering microscopy and microspectroscopy. LANTCET
method was tested <i>in vitro</i> for living cell samples with: (1) model myeloid K562 cells (CD33 positive), (2) primary
human bone marrow CD33-positive blast cells from patients with the diagnosis of acute myeloid leukemia, (3)
monolayers of living EGF-positive carcinoma cells (Hep-2C), (4) human lymphocytes and red blood cells as normal
cells. The LANTCET method was also tested <i>in vivo</i> using rats with experimental polymorphic sarcoma. Photothermal
bubbles were generated and detected <i>in vitro</i> with a photothermal microscope equipped with a tunable Ti-Sa pulsed
laser. We have found that cluster formation caused an almost 100-fold decrease in the bubble generation threshold of
laser pulse fluence in tumor cells compared to the bubble generation threshold for normal cells. The animal tumor that
was treated with a single laser pulse showed a necrotic area of diameter close to the pump laser beam diameter and a
depth of 1-2 mm. Cell level selectivity of tumor damage with single laser pulse was demonstrated. Combining lightscattering
imaging with bubble imaging, we introduced a new image-guided mode of the LANTCET operation for screening and treatment of tumors <i>ex vivo</i> and <i>in vivo</i>.
The ability to detect optical signals form a cellular target depends upon the amount of optical energy that can be
generated by this target as the signal. Given that the sensitivity of optical detectors has some finite limit, further increase
of the sensitivity of optical diagnostic methods requires increasing the energy of target-generated signal. Usually this
energy is converted by the cellular target upon its optical excitation and is limited by many factors such as: cell and
target damage thresholds, efficiency of excitation energy conversion, size of the target etc. All these put principal
limitation on sensing small targets (like molecules) in living cells with any optical method because the energy that can
be safely converted by the target into a signal is limited. To overcome this limitation and to improve the sensitivity of
optical microscopy of living cells (and cytometry in general) we propose the concept of intracellular amplification of the
optical signal. This concept includes two major steps. First, primary (pump) optical radiation interacts with the target (a
probe molecule) to generate a transient target. Second, the transient target is sensed with additional optical radiation that
does not interact strongly with primary target or the cell, and, hence, may have high enough energy to increase the
signal from transient target even above the energy of pump radiation, which is limited by cell and target damage
thresholds. We propose to use optical scattering from clusters of gold nanoparticles (the target) that are selectively
formed in specific cells through antibody-receptor interaction and through endocytosis. To amplify this optical signal
we propose to generate photothermal bubbles (the transient target) around those clusters. In experiments with water
suspensions and with individual tumor K562 cells we have achieved optical signal amplification in individual cells
(relatively to the scattering signal from intact cells): with gold nanorod intracellular clusters, 14.8 times, with
photothermal bubbles, generated around those clusters, more than 100 times. Those signals were much higher than
corresponding fluorescent signals and were obtained from living cells.
Laser Activated Nano-Thermolysis was recently proposed for selective damage of individual target (cancer) cells by
pulsed laser induced microbubbles around superheated clusters of optically absorbing nanoparticles (NP). One of the
clinical applications of this technology is the elimination of residual tumor cells from human blood and bone marrow.
Clinical standards for the safety and efficacy of such procedure require the development and verification of highly
selective and controllable mechanisms of cell killing. Our previous experiments showed that laser-induced microbubble
is the main damaging factor in the case cell irradiation by short laser pulses above the threshold. Our current aim was to
study the cell damage mechanisms and analyze selectivity and efficacy of cell damage as a function of NP parameters,
NP-cell interaction conditions, and conditions of bubble generation around NP and NP clusters in cells. Generation of
laser-induced bubbles around gold NP with diameters 10-250 nm was studied in Acute Myeloblast Leukemia (AML)
cultures, normal stem and model K562 human cells. Short laser pulses (10 ns, 532 nm) were applied to those cells in
vitro and the processes in cells were investigated with photothermal, fluorescent and atomic force microscopies and also
with fluorescence flow cytometry. We have found that the best selectivity of cell damage is achieved by (1) forming
large clusters of optically absorbing NP in target cells and (2) irradiating the cells with single laser pulses with the lowest
fluence that can generate microbubble only around large clusters but not around single NP. Laser microbubbles with the
lifetime from 20 ns to 2000 ns generated in individual cells caused damage and lysis of the cellular membrane and
consequently cell death. Laser microbubbles did not damage normal cells around the damaged target (tumor) cell. Laser
irradiation with equal fluence did not cause any damage of cells without accumulated NP clusters.
Successful targeting nanoparticles (NP) to specific cells requires reliable feedback about NP accumulation in cells. This
task is a challenge for all optical methods due the size of NP and diffraction limit of optical devices. We modified
several microscopy-based techniques for imaging and measuring NP in individual cells: photothermal, fluorescent,
electron and atomic-force microscopies and flow cytometry. All those techniques were applied for quantitative analysis
and imaging of interaction of gold NP (10 and 30 nm) with living tumor cells. Based on experimental results we
performed comparison of all methods in terms of sensitivity, speed, sample requirements etc. We have found that
standard microscopes may detect NP and their clusters in individual living cells through imaging NP-related thermal,
fluorescent and other phenomena.
We describe novel ex vivo method for elimination of tumor cells from bone marrow and blood, Laser Activated Nano-Thermolysis for Cell Elimination Technology (LANTCET) and propose this method for purging of transplants during treatment of leukemia. Human leukemic cells derived from real patients with different diagnoses (acute lymphoblastic leukemias) were selectively damaged by LANTCET in the experiments by laser-induced micro-bubbles that emerge inside individual specifically-targeted cells around the clusters of light-absorbing gold nanoparticles. Pretreatment of the transplants with diagnosis-specific primary monoclonal antibodies and gold nano-particles allowed the formation of nanoparticle clusters inside leukemic cells only. Electron microscopy found the nanoparticulate clusters inside the cells. Total (99.9%) elimination of leukemic cells targeted with specific antibodies and nanoparticles was achieved with single 10-ns laser pulses with optical fluence of 0.2 - 1.0 J/cm<sup>2</sup> at the wavelength of 532 nm without significant damage to normal bone marrow cells in the same transplant. All cells were studied for the damage/viability with several control methods after their irradiation by laser pulses. Presented results have proved potential applicability of developed LANTCET technology for efficient and safe purging (cleaning of residual tumor cells) of human bone marrow and blood transplants. Design of extra-corporeal system was proposed that can process the transplant for one patient for less than an hour with parallel detection and counting residual leukemic cells.
Laser-activated micro- and nano-bubbles (LAB) in cells may be used as universal and sensitive probes for measuring properties of individual cells. Such bubbles can be detected and imaged by using microscopy and flow cytometry. LABs in living blood and tumor cells were induced by pulsed (532 nm, 10 ns) laser radiation and were detected by a thermal lens optical method. LAB lifetime and maximal diameter varied, correspondingly, within the ranges 0.02-10 ms and 0.44-100 mm. LAB parameters - thresholds and probabilities - were found to depend upon the physiological state of cells. Specificity and sensitivity of LAB cytometry were increased by using light-absorbing nanoparticles conjugated to specific monoclonal antibodies.
We are developing new diagnostic and therapeutic technologies for leukemia based on selective targeting of leukemia cells with gold nanoparticles and thermomechanical destruction of the tumor cells with laser-induced microbubbles. Clusters of spherical gold nanoparticles that have strong optical absorption of laser pulses at 532 nm served as nucleation sites of vapor microbubbles. The nanoparticles were targeted selectively to leukemia cells using leukemia-specific surface receptors and a set of two monoclonal antibodies. Application of a primary myeloid-specific antibody to tumor cells followed by targeting the cells with 30-nm nanoparticles conjugated with a secondary antibody (IgG) resulted in formation of nanoparticulate clusters due to aggregation of IgGs. Formation of clusters resulted in substantial decrease of the damage threshold for target cells. The results encourage development of Laser Activated Nanothermolysis as a Cell Elimination Therapy (LANCET) for leukemia. The proposed technology can be applied separately or in combination with chemotherapy for killing leukemia cells without damage to other blood cells. Potential applications include initial reduction of concentration of leukemia cells in blood prior to chemotherapy and treatment of residual tumor cells after the chemotherapy. Laser-induced bubbles in individual cells and cell damage were monitored by analyzing profile of photothermal response signals over the entire cell after irradiation with a single 10-ns long laser pulse. Photothermal microscopy was utilized for imaging formation of microbubbles around nanoparticulate clusters.
Photothermal (PT) responses of individual intact cells are studied with a thermal lens dual-laser scheme. A multiparameter model for analysis of PT responses as a function of cell size, structure, and optical properties is suggested and verified experimentally for living cells, red blood cells, lymphocytes, tumor cells (K 562), hepatocytes, and miocytes, by applying pulsed laser radiation at 532 nm for 10-ns duration. PT responses for noninvasive and damaging modes of laser-cell interaction are investigated. It is shown theoretically and experimentally that specific optical and structural features of cells influence the polarity, shape, front, and tail lengths of their PT responses. Common for different cells, features of PT responses are evaluated. It is found that in cells with a highly heterogeneous light-absorbing structure, the PT response of a whole cell differs from that of the local absorbing area. The model suggested allows us to interpret PT responses from single cells and to compare cells in terms of their diameter, degree of spatial heterogeneity of light absorbance, and laser-induced damage thresholds.