Investigation of PLLA/PCL Blends and Paclitaxel Release Profiles

Blends of poly (l-lactide) (PLLA) and poly (ε-caprolactone) (PCL) with and without paclitaxel were prepared via solution casting. DSC analysis as well as SEM analysis of the PLLA/PCL blend solution cast films showed that these blends are all phase separated.%PLLA crystallinity was found to increase with increasing PCL content (up till 15 wt.%). The PCL phase is found to homogeneously disperse in the PLLA matrix as spherical domains where the pore diameters of the PCL domains significantly increased with increasing PCL content. The degradation profiles matched with the slower degrading component PCL rather than PLLA and also increasing PCL content of the blends increased the degradation rate relatively. The increased crystallinity of the PLLA phase with increasing PCL contents confirmed that the degradation occurred through PCL phase. Cell proliferation on PLLA/PCL blends showed that all these blends were suitable for the support of cellular growth. Apoptosis assay with the paclitaxel-loaded PLLA/PCL blends showed an increase in cell death throughout 7 days of incubation where the cell death was increased with increasing PCL contents. This was attributed to the faster release of paclitaxel which was at least partially affected by the faster degradation rate at increasing PCL contents. The paclitaxel release was shown to be degradation controlled in the initial stages followed by a faster diffusion-controlled release in the later stages. These polymer blends were found to be very suitable paclitaxel release agents for which the paclitaxel release times can be altered with the composition of the blend and the film thickness.     

Fine structure and enzymatic degradation of poly[(R)-3-hydroxybutyrate] and stereocomplexed poly(lactide) nanofibers

Fiber morphology and crystalline structure of poly[(R)-3-hydroxybutyrate] (P(3HB)) and stereocomplexed poly(lactide) (PLA) nanofibers were investigated by using scanning and transmission electron microscopies and X-ray and electron diffractions. In the P(3HB) nanofibers spun from less than 1 wt% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solution, planar zigzag conformation (beta-form) as well as 2(1) helix conformation (alpha-form) structure was formed. Based on the electron diffraction measurement of single P(3HB) nanofiber, it was revealed that the molecular chains of P(3HB) align parallel to the fiber direction. From the enzymatic degradation test of P(3HB) nanofiber, it was shown that beta-form molecular chains are degraded more preferentially than alpha-form chains. Stereocomplexed PLA nanofibers were electrospun from 1 wt% poly(l-lactide)/poly(d-lactide) (PLLA/PDLA) solution in HFIP, which contains equal amounts of PLLA and PDLA. While as-spun stereocomplexed PLA nanofiber was amorphous, PLA nanofiber annealed at 100 degrees C contained only racemic crystal. It was supposed that the crystallization behavior of stereocomplexed PLA in the nanofiber is affected by the electrospinning process, which forcibly exerts the strain onto the polymer chains.     

Structural study of poly(L-lactic acid) spherulites

Spherulites of poly(L-lactic acid) (PLLA) and of its blends with atactic poly(3-hydroxybutyrate) (a-PHB, from 10 to 75 wt %) were investigated by microfocus X-ray diffraction using synchrotron radiation. Radial scans in 5 microm steps with 3 microm beam diameter were performed. In PLLA, tens of identical diffraction images were collected. The unit cell a-axis was radially oriented, and the other axes lacked any specific orientation. In contrast, all PLLA/a-PHB blends showed a periodic change of diffraction pattern with increasing distance from the spherulite center. In all cases, the a-axis lay along the radius, while the b- and c-axes rotated about a with a defined periodicity. The unit cell twisting frequency increased with a-PHB content and closely matched the band spacing observed by polarized optical microscopy, which changed from 250 to 60 microm when the amount of a-PHB increased from 10 to 75 wt %. Concomitantly, a gradual broadening of all X-ray reflections was observed.     

Highly efficient reinforcement of poly-L-lactide materials by polymer blending of a thermotropic liquid crystalline polymer

A novel polymer blend system consisting of poly(l-lactide) (PLLA) and a thermotropic liquid crystalline polymer (LCP: an aromatic polyester comprising poly(4-hydroxybenzoate) sequences) was investigated in the presence and absence of a polycabodiimide (PCD). Scanning electron micrographs of the injection-molded polymer blends revealed the formation of fibrous structure of LCP in the PLLA matrix, supporting the efficient toughening. In particular, the LCP fibrils became semimicrometer in diameter in the presence of PCD with which both PLLA and LCP had reacted during the melt blending to form their block and graft copolymers working as compatibilizer. The blend specimens containing LCP in 20-30 wt % were found to hold high dynamic storage-moduli (E') at high temperature. In addition, the E' value of the specimens containing 30 wt % of LCP reached 10.7 GPa at room temperature, being significantly higher than that of PLLA.     

Stereocomplex formation between enantiomeric poly(lactic acid)s. 12. spherulite growth of low-molecular-weight poly(lactic acid)s from the melt

The spherulite growth of stereocomplex crystallites in the blend from low-molecular-weight poly(L-lactide) [i.e., poly(L-lactic acid) (PLLA)] and poly(D-lactide) [i.e., poly(D-lactic acid) (PDLA)] from the melt, together with that of the homocrystallites in pure PLLA and PDLA films, was investigated using polarization optical miscroscopy. The spherulite growth of stereocomplex crystallites occurred at a wider temperature range (相似文献   

Mechanical properties of laser cut poly(L-lactide) micro-specimens: implications for stent design, manufacture, and sterilization

BACKGROUND: The development of endoluminal stents from polymeric materials requires an understanding of the basic mechanical properties of the polymer and the effects of manufacturing and sterilization on those properties. METHODS: Pure poly(L-lactide) (PLLA) and PLLA containing varying amounts of triethylcitrate (TEC) as a plasticizer (5-10-15%) were studied. The specimens were solution-cast and CO2 laser-cut. Specimen dimensions were adapted to the strut size of polymeric vascular stents. The properties of the PLLA micro-specimens were assessed before and after sterilization (EtO cold gas, H2O2-plasma, beta- and gamma-irradiation). Tensile tests, and creep and recovery tests were carried out at 37 degrees C. Additionally the thermal and thermo-mechanical characteristics were investigated using dynamic-mechanical analysis (DMA) and differential scanning calorimetry (DSC). RESULTS: The results showed the dramatic influence of the plasticizer content and sterilization procedure on the mechanical properties of the material. Laser cutting had a lesser effect. Hence the effects of processing and sterilization must not be overlooked in the material selection and design phases of the development process leading to clinical use. Altogether, the results of these studies provide a clearer understanding of the complex interaction between the laser machining process and terminal sterilization on the primary mechanical properties of PLLA and PLLA plasticized with TEC.     

Environmental degradation of polyester blends containing atactic poly(3-hydroxybutyrate). Biodegradation in soil and ecotoxicological impact

The degradation of poly[(R,S)-3-hydroxybutyrate], a-PHB, binary blends with natural PHB (n-PHB) and poly(L-lactic acid), PLLA, respectively, has been investigated in soil. In such a natural environment, a-PHB blend component was found to biodegrade. The degradation of a-PHB-containing blends proceeded faster than that of respective plain n-PHB and PLLA. The molecular weight decrease of the n-PHB component was higher, while the same rate of bioerosion of both components was observed for the a-PHB/n-PHB binary blend. For the a-PHB blend with PLLA, the weight loss was accompanied by blend composition changes and the decrease of a-PHB content. However, the PLLA molecular weight decrease was lower in the blend in comparison with the plain PLLA sample. The increase of the number of microorganisms particularly observed for the soil where binary blends were incubated indicates that microbial degradation of a-PHB takes place. The terrestrial plant growth test (cress and barley) demonstrates no environmental toxicity of the materials studied.     

Hydroxyapatite surface modified by L-lactic acid and its subsequent grafting polymerization of L-lactide

A new method of surface modification of hydroxyapatite nanoparticles (n-HA) by surface grafting reaction of l-lactic acid and ring-opening polymerization of l-lactide (LLA) was developed. Two modified HA nanoparticles were obtained: HA modified by l-lactic acid (l-HA) and HA grafting with poly(l-lactide) (PLLA; p-HA). The modified surface of n-HA was attested by Fourier transformation infrared, (31)P MAS NMR, and thermal gravimetric analysis. The results showed that l-lactic acid could be easily grafted onto the n-HA surface by forming a Ca carboxylate bond and initiated by the hydroxyl group of the grafted l-lactic acid and LLA could be graft-polymerized onto the n-HA surface in the presence of stannous octanoate. The highest grafting amounts of l-lactic acid and PLLA were about 33 and 22 wt %, respectively. The modified HA/PLLA composites showed good mechanical properties and uniform microstructure. The tensile strength and modulus of the p-HA/PLLA composite containing 15 wt % of p-HA were 67 MPa and 2.1 GPa, respectively, while those of the n-HA/PLLA composites were 45 MPa and 1.7 GPa, respectively. The elongation at the break of the l-HA/PLLA composite containing 15 wt % l-HA could reach 44%, in comparison with 6.5% of the n-HA/PLLA composites containing 15 wt % n-HA.     

A new strategy for recycling and preparation of poly(L-lactic acid): hydrolysis in the melt

Poly(L-lactide) [i.e., poly(L-lactic acid) (PLLA)] was hydrolyzed in the melt in high-temperature and high-pressure water at the temperature range of 180-350 degrees C for a period of 30 min, and formation, racemization, and decomposition of lactic acids and molecular weight change of PLLA were investigated. The highest maximum yield of l-lactic acid, ca. 90%, was attained at 250 degrees C in the hydrolysis periods of 10-20 min. Too-high hydrolysis temperatures such as 350 degrees C induce the dramatic racemization and decomposition of formed lactic acids, resulting in decreased maximum yield of L-lactic acid. The hydrolysis of PLLA proceeds homogeneously and randomly via a bulk erosion mechanism. The molecular weight of PLLA decreased exponentially without formation of low-molecular-weight specific peaks originating from crystalline residues. The activation energy for the hydrolysis (deltaE(h)) of PLLA in the melt (180-250 degrees C) was 12.2 kcal x mol(-1), which is lower than 20.0 kcal x mol(-1) for PLLA and 19.9 kcal x mol(-1) for poly(dl-lactide) [i.e., poly(DL-lactic acid)] as a solid in the temperature range below the glass-transition temperature (21-45 degrees C). This study reveals that hydrolysis of PLLA in the melt is an effective and simple method to obtain l-lactic acid and to prepare PLLA having different molecular weights without containing the specific low-molecular-weight chains, because of the removal of the effect caused by crystalline residues.     

In vivo study on the histocompatibility and degradation behavior of biodegradable poly(trimethylene carbonate-co-D,L-lactide)

The aim of this study was to explore the in vivo behavior and histocompatibility of poly(trimethylene carbonate-co-D,L-lactide) (PDLLA/TMC) and its feasibility of manufacturing cardiovascular stents. Copolymers with 50/50 molar ratio were synthesized by ring-opening polymerization with TMC and D, L-LA, or TMC and L-LA. Poly(L-lactide) (PLLA) was synthesized as a control. The films of the three polymers were implanted into 144 Wistar rats. At different time points of implantation, polymer films were explanted for the evaluation of degradation characteristics and histocompatibility using size exclusion chromatography , nuclear magnetic resonance , environmental scanning electron microscope , and optical microscope. Results showed that there were differences in the percentage of mass loss, molecular weight, shape and appearance changes, and inflammation cell counts between different polymers. With the time extended, the film's superficial structure transformed variously, which was rather obvious in the polymer of PDLLA/TMC. In addition, there were relatively lower inflammation cell counts in the PDLLA/TMC and poly(trimethylene carbonate-co-L-lactide) (PLLA/TMC) groups at different time points in comparison with those in the PLLA group. The differences were of statistical significance (P< 0.05) in the group of PDLLA/TMC vs. PLLA, and the group of PLLA/TMC vs. PLLA, but not within the PDLLA/TMC and PLLA/TMC groups (P> 0.05). These results suggested that the polymer of PDLLA/TMC (50/50) with favorable degradation performance and histocompatibility is fully biodegradable and suitable for manufacturing implanted cardiovascular stents.     

Physical properties, crystallization, and spherulite growth of linear and 3-arm poly(L-lactide)s

The physical properties, crystallization, and spherulite growth behavior and mechanism of linear and 3-arm poly(L-lactide) [i.e., poly(L-lactic acid) (PLLA)] have been investigated using absolute molecular weight as a molecular index. The branching reduces the chain mobility of PLLA and must be excluded from the crystalline regions. The former factor gives the higher glass transition temperature (T(g)) and starting temperature for thermal degradation (T(d,S)) of 3-arm PLLA compared with those of linear PLLA. On the other hand, both the former and the latter factors lead to the higher cold crystallization temperature (T(cc)), the longer induction period for spherulite growth (t(i)), the lower melting temperature (T(m)), crystallinitiy (X(c)), and radius growth rate of the spherulties (G) for the 3-arm PLLA compared with those for the linear PLLA. The G of 3-arm PLLA showed the vague dependence on number-average molecular weight (M(n)), probably because the branching effect was balanced with the molecular weight effect. At the M(n) exceeding critical values, the linear and 3-arm PLLA crystallize in regime II or regime III kinetics, depending on crystallization temperature (T(c)). In contrast, at the M(n) below critical values, the linear and 3-arm PLLA crystallize according solely to regime III and regime II kinetics, respectively, for all the T(c).     

New asymmetric AB(n)-shaped amphiphilic poly(ethylene glycol)-b-[poly(l-lactide)](n) (n = 2, 4, 8) bridged with dendritic ester linkages: I. Syntheses and their characterization

This study presents new investigations on chemical syntheses and characterization of new asymmetric AB(n)-shaped amphiphilic diblock methoxy poly(ethylene glycol)-b-[poly(l-lactide)](n), MPEG-b-(PLLA)(n) (n = 2, 4, and 8), bridged with dendritic ester linkages. First, a new series of A(OH)(n)-shaped hydroxy end-capped MPEG-(OH)(2), MPEG-(OH)(4), and MPEG-(OH)(8) bearing corresponding one- to three-generation dendritic ester moieties were efficiently derived from the starting MPEG (M(n) = 2 KDa) and 2,2'-bis(hydroxymethyl)propionic acid (Bis-HMPA) via ester coupling and a facile hydroxy protection-deprotection cycle, and then, chemical structures of these functional MPEG-(OH)(n) were characterized by nuclear magnetic resonance spectrometry (NMR) and MALDI-FTMS. Subsequently, by employing these MPEG-(OH)(n) as functional macroinitiators, new asymmetric AB(n)()-shaped amphiphilic MPEG-b-(PLLA)(2) S1, MPEG-b-(PLLA)(4) S2, and MPEG-b-(PLLA)(8) S3 bridged with dendritic Bis-HMPA ester linkages of L1-L3 as well as linear structural MPEG-b-PLLA references (R1-R3) were synthesized through the SnOct(2)-catalyzed ring-opening polymerization (ROP) of l-lactide at 130 degrees C in m-xylene solution, and their structures were further examined by NMR and gel permeation chromatography (GPC). It was demonstrated that the functional MPEG-(OH)(n) efficiently initiated the ROP of LLA, finally leading to successful formation of the AB(n)-shaped amphiphilic MPEG-b-(PLLA)(n) (n = 2, 4, and 8) with each PLLA arm weight close to 2 KDa and very narrow molecular weight distribution. Moreover, thermal history, crystallization, and spherulite morphologies were studied by means of differential scanning calorimeter (DSC), thermal gravimetric analyzer (TGA), and polarized microscope (POM) for these new structural amphiphilic S1-S3 as well as the linear R1-R3, intriguingly indicating a strong molecular architecture dependence of segmental crystallizability, spherulite morphology, and apparent crystal growth rate. Due to the favorable biodegradability and biocompatibility of the PLLA and MPEG, these results may therefore create new possibilities for these novel structural AB(n)-shaped amphiphilic MPEG-b-(PLLA)(n) as potential biomaterials.     

Fusidic and helvolic acid inhibition of elongation factor 2 from the archaeon Sulfolobus solfataricus

Fusidic acid (FA) and helvolic acid (HA) belong to a small family of naturally occurring steroidal antibiotics known as fusidanes. FA was studied for its ability to alter the biochemical properties supported by elongation factor 2 isolated from the archaeon Sulfolobus solfataricus (SsEF-2). Both poly(Phe) synthesis and ribosome-dependent GTPase (GTPase(r)) were progressively impaired by increasing concentrations of FA up to 1 mM, whereas no effect was measured in the intrinsic GTPase of SsEF-2 triggered by ethylene glycol in the presence of barium chloride (GTPase(g)). The highest antibiotic concentration caused inhibition of either poly(Phe) synthesis or GTPase(r) only slightly above 50%. A greater response of SsEF-2 was observed when HA was used instead of FA. HA caused even a weak impairment of GTPase(g). A mutated form of SsEF-2 carrying the L452R substitution exhibited an increased sensitivity to fusidane inhibition in either poly(Phe) synthesis or GTPase(r). Furthermore, both FA and HA were able to cause impairment of GTPase(g). The antibiotic concentrations leading to 50% inhibition (IC(50)) indicate that increased fusidane responsiveness due to the use of HA or the L452R amino acid replacement is mutually independent. However, their combined effect decreased the IC(50) up to 0.1 mM. Despite the difficulties in reaching complete inhibition of the translocation process in S. solfataricus, these findings suggest that fusidane sensibility is partially maintained in the archaeon S. solfataricus. Therefore, it is likely that SsEF-2 harbors the structural requirements for forming complexes with fusidane antibiotics. This hypothesis is further evidenced by the observed low level of impairment of GTPase(g), a finding suggesting a weak direct interaction between the archaeal factor and fusidanes even in the absence of the ribosome. However, the ribosome remains essential for the sensitivity of SsEF-2 toward fusidane antibiotics.     

Uniaxial drawing and mechanical properties of poly[(R)-3-hydroxybutyrate]/poly(L-lactic acid) blends

Blends of poly(L-lactic acid) (PLLA) with two kinds of poly[(R)-3-hydroxybutyrate] (PHB) having different molecular weights, commercial-grade bacterial PHB (bacterial-PHB) and ultrahigh molecular weight PHB (UHMW-PHB), were prepared by the solvent-casting method and uniaxially drawn at two drawing temperatures, around PHB's T(g) (2 degrees C) for PHB-rich blends and around PLLA's T(g) (60 degrees C) for PLLA-rich blends. Differential scanning calorimetry analysis showed that this system was immiscible over the entire composition range. Mechanical properties of all of the samples were improved in proportion to the draw ratio. Although PLLA domains in bacterial-PHB-rich blends remained almost unstretched during cold drawing, a good interfacial adhesion between two polymers and the reinforcing role of PLLA components led to enhanced mechanical properties proportionally to the PLLA content at the same draw ratio. On the contrary, in the case of UHMW-PHB-rich blends, the minor component PLLA was found to be also oriented by cold drawing in ice water due to an increase in the interfacial entanglements caused by the very long chain length of the matrix polymer. As a result, their mechanical properties were considerably improved with increasing PLLA content compared with the bacterial-PHB system. Scanning electron microscopy observations on the surface and cross-section revealed that a layered structure with uniformly oriented microporous in the interior was obtained by selectively removal of PLLA component after simple alkaline treatment.     

Comparison of the hydrolytic degradation and deformation properties of a PLLA-lauric acid based family of biomaterials

Addition of lauric acid to PLLA results in a significantly increased rate of hydrolytic degradation, with the time-to-loss of tensile strength directly related to the concentration of lauric acid. In this study, the hydrolytic degradation profiles of four materials were studied: amorphous PLLA, amorphous PLLA containing 1.8 wt % lauric acid, amorphous PLLA containing 4.5 wt % lauric acid, and pre-crystallized PLLA containing 1.8 wt % lauric acid. Hydrolytic degradation was monitored through mass profiles, molecular weight profiles, crystallinity and the development of mechanical properties and deformation mechanisms (through simultaneous small-angle X-ray scattering and tensile testing), and a "phase diagram" of properties suggested. The key factor in determining the development of properties was found to be the time at which crystallization occurred in relation to the loss of molecular weight, with the two factors most affecting this being the lauric acid content and the pre-degradation annealing treatment.     

In-situ formation of biodegradable hydrogels by stereocomplexation of PEG-(PLLA)8 and PEG-(PDLA)8 star block copolymers

Eight-arm poly(ethylene glycol)-poly(L-lactide), PEG-(PLLA)(8), and poly(ethylene glycol)-poly(D-lactide), PEG-(PDLA)(8), star block copolymers were synthesized by ring-opening polymerization of either L-lactide or D-lactide at room temperature in the presence of a single-site ethylzinc complex and 8-arm PEG (M(n) = 21.8 x 10(3) or 43.5 x 10(3)) as a catalyst and initiator, respectively. High lactide conversions (>95%) and well-defined copolymers with PLLA or PDLA blocks of the desired molecular weights were obtained. Star block copolymers were water-soluble when the number of lactyl units per poly(lactide) (PLA) block did not exceed 14 and 17 for PEG21800-(PLA)(8) and PEG43500-(PLA)(8), respectively. PEG-(PLA)(8) stereocomplexed hydrogels were prepared by mixing aqueous solutions with equimolar amounts of PEG-(PLLA)(8) and PEG-(PDLA)(8) in a polymer concentration range of 5-25 w/v % for PEG21800-(PLA)(8) star block copolymers and of 6-8 w/v % for PEG43500-(PLA)(8) star block copolymers. The gelation is driven by stereocomplexation of the PLLA and PDLA blocks, as confirmed by wide-angle X-ray scattering experiments. The stereocomplexed hydrogels were stable in a range from 10 to 70 degrees C, depending on their aqueous concentration and the PLA block length. Stereocomplexed hydrogels at 10 w/v % polymer concentration showed larger hydrophilic and hydrophobic domains as compared to 10 w/v % single enantiomer solutions, as determined by cryo-TEM. Correspondingly, dynamic light scattering showed that 1 w/v % solutions containing both PEG-(PLLA)(8) and PEG-(PDLA)(8) have larger "micelles" as compared to 1 w/v % single enantiomer solutions. With increasing polymer concentration and PLLA and PDLA block length, the storage modulus of the stereocomplexed hydrogels increases and the gelation time decreases. Stereocomplexed hydrogels with high storage moduli (up to 14 kPa) could be obtained at 37 degrees C in PBS. These stereocomplexed hydrogels are promising for use in biomedical applications, including drug delivery and tissue engineering, because they are biodegradable and the in-situ formation allows for easy immobilization of drugs and cells.     

Adsorption characteristics of P(3HB) depolymerase as evaluated by surface plasmon resonance and atomic force microscopy

Molecular recognition of poly[(R)-3-hydroxybutyrate] (P(3HB)) depolymerase from Ralstonia pickettii T1 to the surfaces of biodegradable aliphatic polyesters such as P(3HB) and poly(L-lactic acid) (PLLA) was examined from the viewpoints of kinetics and dynamics. To determine the kinetic parameters on the interaction between the substrate-binding domain (SBD) of P(3HB) depolymerase and various polymer substrates with different chemical structures, surface plasmon resonance (SPR) measurements were performed. On the other hand, using an atomic force microscopic (AFM) cantilever tip functionalized with the SBD of P(3HB) depolymerase, the mechanical parameters such as unbinding force to the polymer surfaces were measured. Both the SPR and AFM measurements showed that the SBD has a high affinity to P(3HB) and PLLA. From the results of kinetics and dynamics, the energy potential landscape of SBD-polymer interaction was disclosed on the basis of a phenomenological model, and the mechanism of the interaction was discussed.     

Desulfurization of Dibenzothiophene and Diesel Oils by a Newly Isolated Gordona Strain, CYKS1

A dibenzothiophene (DBT)-desulfurizing bacterial strain was isolated and identified as Gordona strain CYKS1. Strain CYKS1 was found to transform DBT to 2-hydroxybiphenyl via the 4S pathway and to be able to also use organic sulfur compounds other than DBT as a sole sulfur source. Its desulfurization activity was susceptible to sulfate repression. Active resting cells for desulfurization could be prepared only in the early growth phase. When two types of diesel oils, middle distillate unit feed (MDUF) and light gas oil (LGO) containing various organic sulfur compounds including DBT, were treated with resting cells of strain CYKS1 for 12 h, the total sulfur content significantly decreased, from 0.15% (wt/wt) to 0.06% (wt/wt) for MDUF and from 0.3% (wt/wt) to 0.25% (wt/wt) for LGO. The newly isolated strain CYKS1 is considered to have good potential for application in the biodesulfurization of fossil fuels.     

Production of Phytophthora infestans oospores in planta and inoculum potential of in vitro produced oospores under temperate highlands and subtropical plains of India

High moisture content of the host tissue ( 88%) and low ambient r.h. (50-54%) favoured oospore formation under controlled environments. It took 14–16 days for oospores to develop; thereafter the number of oospores increased with time and decreased with moisture content of host tissue. High ambient r.h. (> 80%) did not favour oospore formation under field or controlled conditions. Oospore formation was detected in inoculated plants grown in the field when the ambient r.h. declined to 74% and moisture content of host tissue decreased to 83.7–85.6%. It took 8 days (cv. Kufri Chandramukhi) to 13 days (cv. Kufri Jyoti and Kufri Badshah) for oospores to develop. Cultivars also differed in their response to oospore production, cv. Kufri Chandramukhi being more responsive (4800 oospores g⁻¹ f wt) than cv. Kufri Jyoti and Kufri Badshah (1320 and 390 oospores g⁻¹ f wt respectively). Oospores produced in vitro remained viable when buried in soil in the temperate highlands of Himachal Pradesh and sub-tropical plains of Uttar Pradesh, India for more than 150 days, i.e. beginning of the next crop season. The oospores germinated and initiated late blight infection at the base of the stems after 21–30 days of incubation of the potato plants raised in oospore-infested soil. It took 2 days for newly formed oospores to germinate and this delay time increased to 75–77 days after 180-days burial. It took 15 days for their germination (47%) in soil extract as compared to 50 days in sterilised distilled water.